TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
112, 198-206
( 1992)
Development of Tolerance to 2-Butoxyethanol-Induced Hemolytic Anemia and Studies to Elucidate the Underlying Mechanisms BURHAN
I. GHANAYEM,’
IDALIA
M. SANCHEZ, AND H. B. MATTHEWS
National Institute of Environmental Health Sciences, National Toxicology Program, Experimental Toxicology Branch, Research Triangle Park, North Carolina 27709 Received July 10, 199 I ; accepted October 23, 199 1
caused by modification of BE metabolism in rats repetitively exposed to this chemical. In conclusion, chronic exposure to BE would be expected to result in tolerance to BE-induced hemolytic anemia. The mechanisms responsible are likely related to the fact that older cells are more susceptible to BE and BAA and that hemolysis of these cells during the initial exposure followed by their replacement with less susceptible younger cells may account for tolerance development. 0 1992 Academic PMS, h.
Development of Tolerance to 2-Butoxyethanol-Induced Hemolytic Anemia and Studies to Elucidate the Underlying Mechanisms. GHANAYEM, B. I., SANCHEZ, I. M., AND MATTHEWS, H. B. (1992). Toxicol. Appl. Pharmacol. 112, 198-206. Early work demonstrated that a single administration of 2butoxyethanol (BE) causes acute hemolytic anemia in rats. Current studies were undertaken to investigate the effect of repetitive daily dosing of BE on the hematologic parameters of male F344 rats. Treatment of rats with BE daily (125 mg/kg/day) for 1 to 3 consecutive days resulted in a time-dependent increase in the hemolysis of erythrocytes. However, when daily treatment with BE continued beyond 3 days, the number of erythrocytes began to rebound and approached pretreatment levels within 12 days despite continued daily exposure, suggesting development of tolerance to the hemolytic effect of BE. In viva and in vitro studies were designed to investigate the underlying mechanism(s) of tolerance to the hematotoxicity of BE. Rats were treated with 125 mg BE/kg/day for 3 days followed by a 7-day recovery. At the end of this recovery period, rats were challenged with a single 125 or 250 mg BE/kg dose and the hematologic profiles were assessed at 2, 8, and 24 hr later. A significant decline in the sensitivity of BE-pretreated/recovered rats compared to vehiclepretreated rats was observed. Further, in vitro incubation of blood obtained from BE-pretreated/recovered with the hematotoxic metabolite of BE, 2-butoxyacetic acid (BAA), revealed that erythrocytes obtained from these rats were significantly less sensitive to BAA than those obtained from normal rats. These studies suggested that tolerance is due, at least in part, to the lesser sensitivity of young erythrocytes formed during the regeneration process. In another study, rats were rendered anemic by bleeding followed by a 7-day recovery. BE administration to bled/recovered rats demonstrated that these rats were less sensitive than rats which were not subjected to bleeding. In vitro incubation of blood obtained from the bled/recovered animals with BAA demonstrated that erythrocytes were significantly less sensitive to BAA than those obtained from control rats. This further conlirmed that young erythrocytes, formed during the regeneration process, were less sensitive to BAA than older erythrocytes. Current data also suggested that it is unlikely that tolerance is ’ To whom
correspondence
should
2-Butoxyethanol (BE), also known as ethylene glycol monobutyl ether, is a major environmental chemical which is utilized in the manufacturing of a wide range of domestic and industrial products. The 1988 annual U.S. production of BE was estimated at over 400 million pounds (ITC, 1988). Administration of a single dose of BE to laboratory rats causes dose-dependent acute hemolytic anemia which is characterized by a decrease in the number of circulating red blood cells, hemoglobin concentration, and hematocrit (Carpenter et al., 1956; Bartnick et al., 1987; Ghanayem et al., 1987a). Furthermore, BE-induced hemolytic anemia was associated with secondary hemoglobinmia and an increase in the spleen wt/body wt ratio (Ghanayem et al., 1987a). In viva and in vitro studies conducted to elucidate the mechanism(s) of BEinduced effects on erythrocytes demonstrated that hemolysis is preceded by swelling [increased hematocrits (HCT) and mean cell volumes (MCV)] and blood ATP depletion (Ghanayem et al., 1990; Ghanayem, 1989). However, it remains unclear if swelling or ATP depletion is the primary effect of BE. In another study by Grant et al. (1985), BE was administered at 500 or 1000 mg/kg/day for up to 22 consecutive days. BE caused hemolytic anemia, splenic extramedullary hemopioesis, hyperplasia of both spleen and bone marrow, and reticulocytosis. Although we reported that human erythrocytes were significantly less sensitive to 2-butoxyacetic acid (BAA) than rat erythrocytes in vitro (Ghanayem, 1989), another report indicated that hemolysis of erythrocytes occurred in a human who ingested a window cleaning solution containing BE (Rambourg-Scherpens et al., 1988).
he addressed.
0041-008X/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
198
TOLERANCE
TO 2-BUTOXYETHANOL
Work performed in this laboratory and others revealed that BE is metabolized to BAA, BE-glucuronide and BEsulfate conjugates (Ghanayem et al., 1987b), and ethylene glycol (Medinsky et al., 1990). Investigation of the metabolic basis of BE-induced hemolytic anemia in rats showed that metabolic activation of BE to BAA via the alcohol and aldehyde dehydrogenases is a prerequisite for the development of hemolytic anemia and BAA is the proximate hematotoxic metabolite (Ghanayem et al., 1987a; Ghanayem, 1989). The initial objective of the current studies was to investigate the effect of daily dosing of male F344 rats with BE for up to 12 days. These studies showed that daily dosing of rats with BE resulted in tolerance development to BE-induced hemolytic anemia. Subsequently, additional in vivo and in vitro studies were undertaken to investigate the mechanism(s) involved in the development of tolerance to the hemolytic effects of BE. MATERIALS
AND METHODS
Chemicals. BE of >99% purity was purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-( l-[14C] Butoxy)ethanol was purchased from New England Nuclear (Boston, MA). The specific activity of BE was 6.32 mCi/ nmol with greater than 99% radiochemical purity. BAA was synthesized as previously described (Ghanayem ef al., 1989). Animafs and treatments. Male Fischer 344 rats (lo- 14 week) were obtained from Charles River Breeding Laboratories (Raleigh, NC), maintained on NIH 31 diet and water ad libitum, and allowed a minimum of 1 week adjustment to our facilities (12-hr dark/light cycle, 2 l-23°C ambient temperature, and a 40-60% relative humidity) prior to inclusion in the present studies. BE dosing solutions were prepared by mixing with water to obtain a dose volume of 5 ml/kg and administered to rats by gavage. Repeat exposure of rats lo BE. At an age of 10-14 weeks, rats were randomly divided into six groups (six rats/group) and treated by gavage as follows: -Group 1 received 5 ml water/kg body wt (BW) daily for 12 consecutive days (control); -Group 2 received 125 mg BE in 5 ml water/kg BW daily for 12 consecutive days; -Group 3 received 125 mg BE in 5 ml water/kg BW daily for 6 consecutive days; -Group 4 received 125 mg BE in 5 ml water/kg BW daily for 3 consecutive days; -Group 5 received 125 mg BE in 5 ml water/kg BW daily for 2 consecutive days; and -Group 6 received a single dose of 125 mg BE in 5 ml water/kg BW. Twenty-four hours after the last dose, rats were anesthetized in a CO2 chamber and two blood samples were collected from the retroorbital venous plexus. One sample was collected into a 0.75-ml capacity Microvettes KE containing 1.5 mg EDTAK2 as anticoagulant. The second blood sample was collected from the same animals using acetate:citrate:dextrose (ACD) solution as anticoagulant (blood:ACD ratio is 4: 1) to be used for the determination of blood ATP concentration. After blood collection, rats were killed by placement in a COZ chamber and the spleen and liver were removed free of other tissues and weighed. Organ weight/ 100 g body wt ratios were calculated and presented as such. Blood samples were analyzed using an Hl hematology analyzer (Technicon, Tarrytown, NY) and the following pa-
HEMATOTOXICITY
199
rameters were measured: red blood cells (RBC) and white blood cell counts, hemoglobin concentrations (HGB), HCT, MCV, mean cell hemoglobin, mean cell hemoglobin concentration, and platelet counts. In addition, packed cell volumes (PCV) were determined on all blood samples. Only parameters which were affected by BE are presented. Blood ATP concentrations were determined in blood samples containing ACD (anticoagulant) as follows: Packed cell volume was immediately determined and blood samples were mixed with trichloroacetic acid, vortexed, and left in an ice cold water bath for 10 min. Samples were centrifuged at 3000g for 10 min and the supematant was aspirated and used for spectrophotometric ATP determination as previously described (Ghanayem, 1989). Hematotoxicity of BE in BE-pretreated/recovered rats in vivo. Rats (lo14 week) were randomized into two groups. Group A received 125 mg BE/ kg/day for 3 consecutive days by gavage (BE-pretreated/recovered). Group B received water, the vehicle, for the same time period. Animals in both groups were then allowed to recover without treatment for 7 days. Groups A and B are refered to as BE-pretreated/recovered and untreated rats, respectively. BE-pretreated/recovered and untreated rats were divided into three subgroups each and treated as follows: subgroup 1 received 5 ml water/ kg (vehicle); subgroup 2 received 125 mg BE/5 ml water/kg; and subgroup 3 received 250 mg BE/5 ml water/kg. Three to five rats from each of the vehicle, low, and high BE dose subgroups were anesthetized in a CO* chamber at 2. 8, or 24 hr after dosing, and two blood samples were collected from the retroorbital venous plexus. One blood sample was collected using EDTA as an anticoagulant and analyzed using an H 1 hematology analyzer as described above. The second blood sample was collected using ACD as an anticoagulant and utilized for the determination of PCV and ATP concentration in blood. After blood collection, rats were killed by placement in a COZ chamber and the spleen was removed and weighed. The spleen wt/lOO g body wt was calculated and presented as such. Effect ofBAA on bloodfrom BE-pretreated/recovered rats in vitro. Rats were treated with 125 mg BE/kg/day for 3 consecutive days and allowed a 7-day recovery (BE-pretreated/recovered). Blood was collected from CO2 anesthetized rats (>5) by cardiac puncture using ACD as an anticoagulant and pooled. Blood was incubated with BAA at a final concentration of 0, 0.5, 1.0, or 2.0 mM. Samples were incubated at 37°C for 0.5, 1.0, 2.0, or 4.0 hr. At the end of the incubation period, PCV and blood ATP concentrations were measured as described above. Hematotoxicity of BE in bled/recovered rats in vivo. Rats ( IO- I4 week) were lightly anesthetized in a CO2 chamber, bled (4-5 ml blood/rat) via the retroorbital venous plexus, and were allowed a 7day holding period to permit the recovery of the hematopoietic system (bled/recovered rats). Rats were randomized into three groups. Group A received water, the vehicle; group B received a single dose of 125 mg BE/kg BW by gavage; and group C received a single dose of 250 mg BE/kg BW by gavage. Three to four rats from each of the vehicle, low, and high BE dose groups were anesthetized in a CO2 chamber at 2,8, or 24 hr after dosing; a blood sample was collected from the retroorbital venous plexus using EDTA as an anticoagulant and analyzed using an H 1 hematology analyzer as described above. After blood collection, rats were euthanized by placement in a COZ chamber and the spleen was removed and weighed. The spleen wt/lOO g body wt. was calculated and presented as such. Effect of BAA on blood from bled/recovered rats in vitro. Rats were lightly anesthetized in a CO2 chamber and bled (4-5 ml blood/rat) via the retroorbital venous plexus. Animals were allowed a 7-day holding period to allow for the recovery of the hematopoietic system. Using cardiac puncture of CO2 anesthetized animals, blood was collected using ACD as an anticoagulant. Blood was pooled and the effect of BAA on PCV and ATP concentration was investigated as outlined in the previous section. Eflect of multiple dosing of rats with BE on its own disposition and metabolism. Rats were treated with 125 mg/kg BW/day of unlabeled BE for 3 or 7 days by gavage followed by a single dose of 125 mg/kg BW of r4CBE (40-50 &i/kg) on Days 4 and 8. respectively. Another untreated three
200
GHANAYEM,
0 (control) 1 2 3 6 12
AND MATTHEWS
TABLE 1 Parameters” of Male F344 Rats Treated with 2-Butoxyethanolb Daily for Various Periods of Time
Hematologic Days of treatment
SANCHEZ,
RBC (106/d) 8.9 5.1 4.2 4.4 5.9 6.8
+ * + f + f
HGB Wl)
0.2 0.2* 0.4* 0.2* 0.1* 0.1*
15.3 iz 0.2 8.7 5 0.2* 7.2 f 0.6* 8.0 f 0.3* 11.2 f 0.2* 13.0 + 0.1*
HCT 6) 46.8 28.4 24.0 26.1 38.5 43.0
f + + -t + +
MCV (fl)
0.8 0.9* 1.8* 0.9* 0.7* 1.0:
52.5 55.5 56.9 59.5 65.5 63.3
k * + f f f
ATP (rc.mol/g HGB)
0.2 0.5* 0.9* 0.5* 1.6* 0.5*
4.1 4.8 5.9 7.0 7.4 5.3
Recticulocytes (million/~l)
+ 1.0 k 1.2; f 1.9* iz 4.6* rf: 1.7* + 0.8*
0.14 0.23 0.26 0.36 0.87 0.36
k + + f f f
0.05 0.06* 0.03* 0.02* 0.14* 0.06*
a Data are presented as the mean _+ SE of four to five rats. * 2-Butoxyethanol was administered daily at 125 mg/kg/day and rats were killed 24 hr after the last dose. * Significantly different from control at d< 0.05. - -’ -
rats received the same dose of 14C-BE. Rats treated with radiolabeled BE were immediately placed in glass metabolism cages which allowed separate collection of fecesand urine. Expired volatiles and 14C02were also collected. Disposition and urine analysis of BE metabolites were performed as previously described (Ghanayem et al., 1987b). Statisticul analysis. Statistical analysis was performed using a pairwise comparison of variance (pooled t test). Values were considered statistically significant at p < 0.05.
RESULTS Efect of repeat exposure of rats to BE. As observed in earlier studies (Ghanayem et al., 1990) administration of a single dose of BE (125 mg/kg) to rats resulted in significant hemolysis of erythrocytes as evidenced by a decreased RBC, HGB, and HCT (Table 1). This effect became more pronounced following the third day of dosing, but gradual recovery was observed thereafter. With continued repeated exposure to BE beyond Day 3, rats exhibited a gradual increase in RBC and HGB and approached pretreatment levels by Day 12 (Table 1). HCT followed a similar pattern as RBCs and HGB as dosing continued. On the other hand, MCV increased as the dosing regimen was extended to 6 days and declined to remain above control levels throughout the 12day study (Table 1). ATP concentration, MCV, and the number of reticulocytes increased after 6 days and then declined by Day 12 (Table 1). Reticulocytosis and increased ATP concentration were evident after the second day of dosing and persisted in BE-treated rats throughout the study. The effect of repeat dosing of rats with BE on the weight of the spleen and liver is shown in Fig. 1. Spleen wt/body wt ratios markedly increased to a maximum after 6 days from which they declined between Days 6 and 12 (Fig. 1). Liver wt/body wt ratios were minimally affected by repeat dosing and, following a moderate decline on Days 3 and 6, they were increased on Day 12 compared to those observed in controls (Fig. 1). Hematologic parameters of BE-pretreated/recovered rats treated with BE in vivo. Comparison of BE-induced he-
matotoxicity in BE-pretreated/recovered and untreated rats revealed that BE-pretreated/recovered rats were significantly less sensitive to the hemolytic effects of BE than untreated rats (rats which were not previously exposed to BE; Table 2). The RBC and HGB declined in a dose- and time-dependent manner, however, there was a minimal decline in these parameters in rats pretreated with the low dose of BE (Table 2). HCT increased at 2 hr and remained near or slightly below control in BE-pretreated/recovered rats. In contrast, rats which were not previously exposed to BE exhibited an increase in HCT early after treatment which later declined in a dose- and time-dependent manner (Table 2). Treatment
160
I \‘\ ‘\ ,I ‘\ ‘\ ‘\
0
2
4
6
6
10
‘\ \ I
12
14
TIME (days)
FIG. 1. Effect of administration of BE (125 mg/kg/day) on the liver (-) and spleen (---) weights/body wt ratios of F344 male rats. Values are presented as percentage control and are the mean + SE of three to six rats.
TOLERANCE
TO 2-BUTOXYETHANOL
201
HEMATOTOXICITY
TABLE 2 Comparison of the Effects of 2-Butoxyethanol” on the Hematologic Parameters of Untreated and BE-Pretreated/Recovered” Male F344 Rats Time (hr)
RBC’
HGB’
HCTC
MCV’
Untreated ( 125 mgfkg)
2 8 24
87.6 zk 1.6 82.9 t 3.9 66.4 f 2.9
88.0 2 1.5 85.4 i- 2.9 65.4 + 2.8
114.3 rt 1.7 92.9 * 2.6 61.3 + 3.8
116.4 + 2.0 101.1 z!z2.3 91.0 + 0.3
59.0 f 2.9 68.3 f 1.5 10.5 f 3.5
0.13 + 0.03 0.21 k 0.08 0.22 * 0.02
BE-Pretreated (125mgk)
2 8 24
95.6 f 0.6 94.6 f 1.3 98.9 -c 0.7
94.8 f 0.2 93.7 f 1.3 98.6 -c I.0
112.6 k 0.6 101.5 z!z0.8 102.3 EL 1.5
117.5 + 0.6 107.3 f 0.6 102.8 -c 1.5
116.1 f 1.4 93.4 f 1.2 105.4 iz 5.1
0.44 + 0.05 0.32 + 0.06 0.50 f 0.07
Untreated (250w/kg)
2 8 24
79.7 f 1.3 58.8 f 1.6 56.1 + 0.7
81.5 + 1.4 85.4 f 2.9 55.5 f 0.7
108.8 + 1.61 13.1 * 2.2 61.4 k 1.5
122.0 + 0.0 111.9?2.9 98.8 + 2.8
39.3 + 2.9 56.9 rf: 2.6 64.9 + 1.7
0.17 + 0.03 0.30 * 0.05 0.30 k 0.02
BE-Pretreated (250 w/W
2 8 24
92.6 + 1.1 91.2 + 1.4 90.6 f 1.1
90.2 + 0.8 91.4 r 0.8 91.6 + 1.8
113.9 + 1.4 106.8 t 2.2 95.00 f 1.2
122.6 + 0.56 118.1 r 2.0 104.5 * 1.5
76.1 zk 3.9 85.3 i 1.5 92.3 ?I 2.4
0.41 + 0.04 0.48 k 0.02 0.50 * 0.03
Treatment
ATP
Recticulocytesd
a 2-Butoxyethanol was administered by gavage at a dose volume of 5 ml/kg. b Rats were treated with 125 mg BE/kg for 3 consecutive days and allowed 7 days recovery. ’ Data are presented as percentage of the corresponding control and are the mean f SE of four to five rats. d Reticulocvtes are presented in millions/u1 of blood. Reticulocvtes in untreated and BE-pretreated controls were 0.13-0.23 and 0.42-0.45 million/r] of blood, respectively.-
of BE-pretreated/recovered rats with the high BE dose (250 mg/kg) caused more pronounced hematotoxicity than the low dose ( 125 mg BE/kg); however, this effect remained significantly less than that observed in untreated rats receiving the same BE dose (Table 2). BE-pretreated/recovered rats were also less sensitive to the increase in MCV and ATP depletion caused by BE than rats which were not previously exposed to BE (Table 2). In addition, the number of reticulocytes was significantly higher in rats pretreated with BE than in untreated controls (Table 2). Comparison of the spleen w/body wt ratio in these rats is shown in Fig. 2 and demonstrated that the increase caused by BE is significantly less in BE-pretreated rats than in untreated rats (Fig. 2). Efect of BAA on blood from BE-pretreated/recovered rats in vitro. Incubation of blood obtained from rats pretreated with BE for 3 consecutive days and allowed a 7-day recovery with the toxic metabolite of BE, BAA, suggested that RBCs are significantly less sensitive to BAA than those obtained from untreated rats (Figs. 3 and 4). Figure 3 shows that the increase in PCV caused by BAA in blood from untreated rats was significantly greater than that caused in blood from BE-pretreated rats. Similarly, the effect of BAA on ATP concentration in blood from both the BE-pretreated/recovered and untreated rats revealed that pretreatment of rats with BE decreased the susceptibility of erythrocytes to the effect of BAA in vitro (Fig. 4). Both effects of BAA became more dramatic as the concentration of BAA and the incubation time increased.
Hematologic parameters of bled/recovered rats treated with BE in vivo. Comparison of the hematotoxicity of BE in bled/recovered and controls (not bled) rats revealed that bled/
0
5
10
15
20
25
TIME (hr)
FIG. 2. Effect of administration of a single 250 (0) and 125 (m) mg BE/ kg dose on the spleen wt/body wt ratio of control (-) and BE-pretreated/ recovered (---) male F344 rats. Values are presented as percentage control and are the mean f SE of three to six rats.
202
GHANAYEM,
SANCHEZ, AND MATTHEWS
160-
g 3
140-
g= -lg d,’ 00 09 ue :: d
130.
120-
110-
0
1.0
2.0
3.0 TIME
4.0
(hr)
FIG. 3. Effect of BAA concentration and time of incubation on packed cell volume of blood obtained from control (-) and BE-pretreated/recovered (---) male F344 rats. Values are presented as percentage control and are the mean + SE of six to nine determinations. BAA final concentrations in blood were: A, 2.0 mM: 0, 1.0 mM; n , 0.5 mM.
recovered rats were significantly less sensitive to the hemolytic effects of BE than controls (Table 3). The RBCs and HGB declined in a dose- and time-dependent manner; however, the decline in bled/recovered rats treated with the 125 mg BE/kg BW was relatively minimal (Table 3). HCT increased at 2 hr and remained near pretreatment levels in bled/recovered rats. In contrast, BE administration to controls resulted in an early increase in HCT and then declined in a dose- and time-dependent manner (Table 3). Although the number of reticulocytes in bled/recovered rats prior to exposure to BE was significantly higher than in the corresponding controls, this trend increased or remained the same after exposure to BE (Table 3). Treatment of bled/recovered rats with the high BE dose (250 mg/kg) caused more pronounced hematotoxicity than the low dose (125 mg/kg); however, these effects continued to be less pronounced than those observed in control rats receiving the same BE dose (Table 3). Comparison of the spleen wt/body wt ratio demonstrated that there was a significantly greater increase caused by BE in control rats compared to bled/recovered rats (data not shown). Efsect of BAA on blood from bled/recovered rats in vitro. Comparison of the effect of BAA on blood from bled/ recovered and normal rats is shown in Figs. 5 and 6. Blood obtained from bled/recovered rats was less sensitive to BAA-
induced increases in PCV and ATP depletion. These effects were more pronounced at higher BAA concentrations and at later incubation times (Figs. 5 and 6). Eflect of multiple dosing of rats with BE on its own disposition and metabolism. The effect of multiple dosing of rats with BE on its own metabolism and disposition is shown in Tables 4 and 5. CO2 exhalation and urinary excretion were shown as the two major routes of BE elimination (Ghanayem et al., 1987b). The percentage of the administered BE dose eliminated as CO1 and in the urine remained unaffected by pretreatment of rats with BE for 4 or 8 days. Seventeen to 20% of a 125 mg BE/kg dose was exhaled as 14C02 by rats receiving 1, 4, or 8 doses of BE. Urinary excretion of BE-derived radioactivity also remained unchanged and ranged from 55 to 65% of the administered dose in 24 hr (Table 4). Results of the HPLC analysis of the urinary metabolites of BE in rats treated with BE for 1, 4, or 8 days are shown in Table 5. No significant difference in the percentage of dose excreted in the urine as BAA (hematotoxic metabolite) was detected between the three groups of rats (Table 5). Although the percentage of dose excreted as BAA in the urine at 8-24 vs O-8 hr has increased within each of the groups, it remained essentially the same in the three groups. In addition, no major differences in the percentage of dose excreted as BE and BE-glucuronide and BE-sulfate
1
30&Y, 0 1.0
I 2.0
I 3.0
4.0
TIME (hr)
FIG. 4. Effect of BAA concentration and time of incubation on ATP concentration in biood obtained from control (-) and BE-pretreated/recovered (---) male F344 rats. Values are presented as percentage control and are the mean f SE of six to nine determinations. BAA concentrations in blood were: A, 2.0 mM; 0, I.0 mM: n , 0.5 mM.
TOLERANCE
TO 2-BUTOXYETHANOL
203
HEMATOTOXICITY
TABLE 3
Comparison of the Effects of 2-Butoxyethanol” on the Hematologic of Control and PrebledlRecovered* Male F344 Rats
Parameters
Time U-4
RBC’
HGB’
HCT’
Control ( 125 mg BE/kg)
2 8 24
88 f 1.6 83 k 3.9 66 k 2.9
88 f 1.5 85 f 2.9 65 f 2.8
114 f 1.7 93 + 2.6 67 k 3.8
116 + 2.0 101 f 2.3 91 f 0.3
0.13 rt 0.03 0.21 + 0.08 0.22 Ik 0.02
Prebled ( 125 mg BE/kg)
2 8 24
94 f 0.9 91 f 3.8 88 f 0.6
91 + 0.6 94 + 2.5 92 + 0.5
115kO.6 105 + 2.5 94 f 0.8
121 + 1.0 116 k 1.1 106 f 1.0
0.54 + 0.0 1 0.66 + 0.04 0.51 * 0.02
Control (250 mg BE/kg)
2 8 24
80 + 1.3 59 + 1.6 56 f 0.7
82 + 1.4 85 + 2.9 56 + 0.7
109 f 1.61 73 f 2.2 61 rl 1.5
122 + 0.0 112 ?I 2.9 99 f 2.8
0.17 f 0.03 0.30 f 0.05 0.30 * 0.02
Prebled (250 mg BE/kg)
2 8 24
88 + 1.5 83 + 2.2 82 + 2.0
87 + 1.5 86 f 1.7 83 f 2.4
114 + 1.5 101 f 4.0 91 f 2.7
130 + 1.7 121 + 3.0 111 + 1.5
0.47 + 0.02 0.63 + 0.06 0.52 + 0.01
Treatment
MCV’
Recticulocytesd
” 2-Butoxyethanol was administered by gavage at a dose volume of 5 ml/kg. b Rats were bled and allowed a ‘I-day recovery prior to BE administration. ’ Data are presented as percentage of the corresponding control and are the mean + SE of three to five rats. d Reticulocytes are presented in million/r1 blood. Reticulocytes in control and bled/recovered rats were 0.13-0.23 and 0.39-0.56 million/r1 blood, respectively.
conjugates were observed in rats treated with BE for 1,4, or 8 days (Table 5). DISCUSSION
Administration of a single dose of BE to laboratory animals caused dose- and time-dependent hemolytic anemia (Carpenter et al., 1956; Ghanayem et al., 1987a). BE-induced hemolytic anemia was characterized by an early swelling of erythrocytes (increased HCT and MCV) associated with ATP depletion, resulting in hemolysis and decline in the number of circulating erythrocytes, hemoglobin concentration, and hematocrit (Ghanayem, 1989; Ghanayem et al., 1990). In addition, BE caused secondary hemoglobinurea and increased spleen wt/body wt ratio (Ghanayem et al., 1987a). Spleen enlargement was attributed to sequestration of swelled/deformed RBCs and was directly related to the effect of BE on RBCs. Current studies were undertaken to investigate the effects of repeat exposure of male F344 rats to a relatively low dose of BE. Results presented in this report indicated that repetitive daily dosing of rats with 125 mg BE/kg/day resulted in the development of hemolytic anemia, which reached a maximum after 2-3 days. Continued administration of the same BE dose beyond the third day resulted in gradual recovery from the anemic condition in association with tolerance development to the hemolytic effect of BE. This tolerance was characterized by a gradual recovery of the hematologic pa-
rameters (increase in RBCs, HGB, HCT, and MCV) despite continued daily exposure to BE. In addition, ATP concentration in blood increased with time to reach a maximum after 6 days of dosing. Reticulocytosis (increased number of reticulocytes in blood) was also evident after the second dose of BE and persisted throughout the 12 days of repeated exposure. Both the number of reticulocytes and the ATP concentration in the blood declined after 12 days compared to that observed after the sixth day (Table 1). Although it was not specifically identified, tolerance to BE-induced hemolytic anemia was also detectable upon close examination of other prechronic studies (Krasavage, 1986; Grant et al., 1985). In these studies, administration of BE at doses as high as 1000 mg/kg/day for 6 weeks resulted in significantly less pronounced hemolytic anemia than we observed 24 hr after a single 250 or 500 mg BE/kg dose (Ghanayem et al., 1990). It is likely that tolerance was not specifically identified in early studies (Krasavage, 1986; Grant et al., 1985) because ~~~mparison of preclironic and acute (the effect of a single BE dose was not included in the studies) effects of BE were not included. In previous studies, we have demonstrated that metabolic activation of BE to BAA via the alcohol and aldehyde dehydrogenases is a prerequisite for hemolysis of erythrocytes in rats receiving BE and that BAA is the proximate hemolytic agent (Ghanayem et al., 1987a). Other metabolites of BE appear to have little effect on RBCs. Therefore, one hypothesis to explain the biological basis of tolerance development
204
GHANAYEM,
SANCHEZ, AND MATTHEWS
160-
1001’ 0
1.0
2.0
3.0
TIME
0
4.0
1.0
has initially centered on the premise that continued administration of BE may have caused a gradual inhibition of BE metabolism to BAA or induction of an alternative metabolic pathway(s). To address this hypothesis, we examined the metabolism of 14C-labeled BE by rats pretreated with unlabeled BE for 4 or 8 consecutive days. These studies revealed that no quantitative or qualitative alterations of BE metabolism and disposition were caused by repeat exposure to BE compared to those observed in rats treated with a single dose. Elimination of 14C02 and BE-derived radioactivity in the urine of rats receiving multiple doses of BE was essentially the same as in rats receiving a single BE dose (Table 4). In addition, the ratios of BAA (hematotoxic metabolite), the glucuronide and sulfate conjugates, and parent BE excreted in the urine of rats treated for 4 or 8 days were relatively similar to those from rats treated with a single BE dose (Table 5). Therefore, it is unlikely that tolerance development to the hemolytic effects of BE is caused by increased detoxification of BE or inhibition of BE metabolism to BAA. It is well known that aging of erythrocytes results in several biochemical and biophysical changes. For example, the concentrations of glutathione, superoxide dismutase, ATP, and 2,3diphosphoglycerate decrease while the concentration of lipid peroxides increases during the aging of erythrocytes (Abraham et al., 1978; Glass and Gershon, 1981; Turner et
1 3.0
TIME
(hr)
FIG. 5. Effect of BAA concentration and time of incubation on packed cell volume of blood obtained from control (-) and previously bled/recovered (---) male F344 rats. Values are presented as percentage control and are the mean + SE of six to nine determinations. BAA concentrations in blood were: A, 2.0 mM; 0, 1.0 mM; n , 0.5 mM.
1 2.0
1 4.0
1
(hr)
FIG. 6. Effect of BAA concentration and time of incubation on ATP concentration in blood obtained from control (-) and previously bled/ recovered (---) male F344 rats. Values are presented as percentage control and are the mean f SE of six to nine determinations. BAA concentrations in blood were: A, 2.0 mM; 0, 1.0 mM; n , 0.5 mM.
al., 1974; Shiga et al., 1979; Glass and Gershon, 1984). In addition, erythrocytes become more osmotically fragile and less deformable during the aging process (Shiga et al., 1979). These data suggest that erythrocytes undergo significant changes during aging which may result in increasing their susceptibility to chemical-induced hemolysis. We therefore hypothesized that there is a direct relationship between the age of erythrocytes and their sensitivity to the hemolytic effect of BE with older erythrocytes being more susceptible. Early in the dosing regimen with BE, the highly sensitive old erythrocytes undergo hemolysis leaving the less sensitive young erythrocytes. The selective hemolysis of older erythrocytes
TABLE 4 Effect of Dosing Rats with BE” for up to 8 Days on Its Own Metabolism
‘TO2 Urine
One dose
Four doses
Eight doses
17.2 + 0.5b
19.8 k 1.3 57.3 f 5.4
20.0 * 1.4 55.1 + 4.0
64.6 f 5.7
0 Unlabeled BE was administered for 3 or 7 days followed by ‘%-BE on Day 4 (four doses) and Day 8 (eight doses). A third group received a single 14C-BE (one dose). b Values are the mean f SE of three animals.
TOLERANCE
TO 2-BUTOXYETHANOL
205
HEMATOTOXICITY
TABLE 5 Comparison of the Effect of Daily Dosing of Rats with BE” for 1,4, or 8 Days on the Excretion of BE Urinary Metabolites Four doses
One dose Metabolitesb BAA BEG BES BE
8 hr 67.8 24.0 3.5 2.2
+- 3.0’ -t 2.0 I? 0.3 + 1.0
24 hr 91.6 k 1.1 6.6 t 1.1 ND ND
24 hr
8 hr 68.5 11.9 5.1 6.8
f + + f
Eight doses
0.7 1.4 0.2 1.1
8 hr
84.2 f 4.8 8.1 f 0.9 ND ND
65.5 24.2 2.4 4.9
+ f + f
24 hr 5.7 5.4 0.3 3.6
90.2 ?I 0.4 5.2 F 1.4 ND ND
a Unlabeled BE was administered for 3 or 7 days followed by 14C-BE on Day 4 (four doses) and day 8 (eight doses). A third group received a single 14CBE (one dose). b BAA, 2-butoxyacetic acid, BEG, glucuronide conjugate of BE; BES, sulfate conjugate of BE. ’ Values are the mean -CSE of three animals.
in association with the development of reticulocytosis during the process of active blood regeneration may lead to tolerance to the hemolytic effects of BE. This hypothesis was addressed by investigating the effect of BE and BAA on erythrocytes of anemic/recovered rats in vivo and in vitro, respectively. Anemia was induced either by treatment of rats with BE (3 consecutive days at 125 mg/kg/day) or by bleeding. These animals were then allowed to recover for 7 days. Results presented in this report demonstrated that anemic/recovered animals were significantly less sensitive to BE than control rats. However, tolerance to BE was more pronounced in BEpretreated/recovered rats than in bled/recovered rats. This discrepancy is very likely related to the fact that bleeding resulted in the loss of RBCs of ah ages while BE-pretreatment resulted in the selective removal (hemolysis) of the sensitive older cells. Furthermore, erythrocytes obtained from anemic/ recovered rats were found to be less sensitive to BAA in vitro than erythrocytes obtained from control rats. These data, especially the effect of BAA in vitro, confirm the conclusion that tolerance to BE is not mediated by changes in BE metabolism and is most likely caused by the increased susceptibility of erythrocytes to BE/BAA as a function of age. Tolerance development to hemolytic agents such as the antimalarial drug primaquine has long been recognized and documented and was thought to be caused by the higher susceptibility of older RBCs vs younger cells (Kellermeyer et al., 1962). Although the mechanism of RBCs hemolysis by BE is not fully understood, it is unlikely that the cellular basis for tolerance development to primaquine and BE is identical. There is evidence which suggests that the pattern and mechanisms of hemolysis caused by both agents are different. For example, BE-induced hemolysis is preceded by swelling of erythrocytes while primaquine-induced hemolysis is not mediated by erythrocytic swelling. In conclusion, tolerance to BE-induced hemolysis developed upon repeated daily exposure of animals to this chemical. The mechanism(s) underlying this phenomena may be
due, at least in part, to the selective hemolysis of older erythrocytes and their replacement during hematopoiesis with less sensitive young cells. However, the specific change(s) in aged erythrocytes which may increase their sensitivity to hemolysis by BE or BAA remain to be investigated. This report further suggests that chronic administration of BE may cause anemia; however, this effect is unlikely to be as severe as that caused by a single dose of BE. Chronic administration is expected to result in severe anemia early on followed by gradual development of tolerance and equilibrium between hematopoiesis and hemolysis of erythrocytes. This equilibrium is expected to be associated with a decrease in the halflife of erythrocytes leading to lower number of erythrocytes in rats treated with BE chronically compared to controls. It is, therefore, expected that chronic administration of BE may cause hematopoiesis to be sustained above the normal rate, which may subject the hematopoietic system to sustained stress with possible adverse consequences on systems such as spleen and bone marrow. ACKNOWLEDGMENT The authors thank Ms. Sandra Ward for her excellent technical help.
REFERENCES Abraham, E. C., Taylor, F. J., and Lang, C. A. (1978). Influence of mouse age and erythrocyte age on glutathione metabolism. Biochem. J. 174, 819-825. Bartnik, F. G., Reddy, A. K., Klecak, G., Zimmermann, V., Hostynek, J. J., and Kunstler, K. (1987). Percutaneous absorption, metabolism, and hemolytic activity of n-butoxyethanol. Fundam. Appl. Toxicol. 8,59-70. Carpenter, P. C., Pozzani, C. U., Weil, C. S., Nair III, H. J., Keck, G. A., and Smyth, H. F. (1956). The toxicity of butyl cellosolve solvent. Arch. Ind. Health
14, 114-131.
Ghanayem, B. I. (1989). Investigation of the mechanisms of 2-butoxyethanol induced hemolytic anemia in rats and assessment of human risk in vitro. Biochem. Pharmacol. 38, 1679-1684. Ghanayem, B. I., Burka, L. T., and Matthews, H. B. (1987a). Metabolic basis of ethylene glycol monobutyl ether (2-butoxyethanol) toxicity: Role
206
GHANAYEM,
SANCHEZ,
of alcohol and aldehyde dehydrogenases. J. Pharmacol. Exp. Ther. 242, 222-231. Ghanayem, B. I., Burka, L. T., Sanders, J. M., and Matthews, H. B. (1987b). Metabolism and disposition of ethylene glycol monobutyl ether (2-butoxyethanol) in rats. Drug Metab. Dispos. 15, 478-484. Ghanayem, B. I., Burka, L. T., and Matthews, H. B. (1989). Structureactivity relationships for the in vitro hematotoxicity of n-alkoxyacetic acids, the toxic metabolites ofglycol ethers. Chem. Biol. Interact. 70,339352. Ghanayem, B. I., Ward, S. M., Blair, P. C., and Matthews, H. B. (1990). Comparison of the hematologic effectsof 2-butoxyethanol using two types of hematology analyzers. Toxicol. Appl. Pharmacol. 106, 341-345. Glass, G. A., and Gemhon, D. (198 1). Enzymatic changes in rat erythrocytes with increasing cell and donor age: Loss of superoxide dismutase activity associatedwith increases in catalytically defective forms. Biochem. Biophys. Res. Commun. 103, 1245-1253. Glass, G. A., and Gershon, D. (1984). Decreased enzymatic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell and donor aging. Biochem J. 218,531-537. Grant, D., Slush, S., Jones, B. W., Gangolli, D. S., and Butler, W. H. (1985). Acute toxicity and recovery in the hematopoietic system of rats after
AND MATTHEWS
treatment with ethylene glycol monomethyl and monobutyl ethers. Tox77, 187-200. ITC/International Trade Commission Report on Annual Production of the Year 1987. September 1988. Kellermeyer, R. W., Tarlov, A. R., Brewer, G. J., Carson, P. E., and Alving, A. S. ( 1962). Hemolytic effect of therapeutic drugs: Clinical considerations of the primaquine-type hemolysis. JAMA 180, 388-394. Krasavage, W. J. (1986). Subchronic oral toxicity of ethylene glycol monobutyl ether in male rats. Fundam. Appt. PharmacoL 6, 349-355. Medinsky, M. A., Singh, G., Bechtold, W. E., Bond, J. A., Sabourin, P. J., Birnbaum, L. S., and Henderson, R. F. (1990). Disposition of three glycol ethers administered in drinking water to male F344/N rats. Toxicol. Appl. icol. Appl. Pharmacol.
Pharmacol.
102,443-445.
Rambourg-Schepens, M. O., Buffet, M., Bertault, R., Jaussaud, M., Joume, B., Fay, R., and Lamiable, D. (1988). Severe ethylene glycol butyl ether poisoning: Kinetics and metabolism pattern. Human Toxicol. 7, 187189. Shiga, T., Maeda, N., Suds, T., Kon, K., and Sekiya, M. (1979). The decreased membrane fluidity of in vivo aged human erythrocytes. Biochem. Biophys. Acta 553, 84-95.
Turner, B. M., Fisher, A. R., and Harris, H. (1974). The age related loss of activity of four enzymes in the human erythrocytes. C/in. Chim. Acta 50, 85-95.
AND
APPLIED
PHARMACOLOGY
112, 198-206
( 1992)
Development of Tolerance to 2-Butoxyethanol-Induced Hemolytic Anemia and Studies to Elucidate the Underlying Mechanisms BURHAN
I. GHANAYEM,’
IDALIA
M. SANCHEZ, AND H. B. MATTHEWS
National Institute of Environmental Health Sciences, National Toxicology Program, Experimental Toxicology Branch, Research Triangle Park, North Carolina 27709 Received July 10, 199 I ; accepted October 23, 199 1
caused by modification of BE metabolism in rats repetitively exposed to this chemical. In conclusion, chronic exposure to BE would be expected to result in tolerance to BE-induced hemolytic anemia. The mechanisms responsible are likely related to the fact that older cells are more susceptible to BE and BAA and that hemolysis of these cells during the initial exposure followed by their replacement with less susceptible younger cells may account for tolerance development. 0 1992 Academic PMS, h.
Development of Tolerance to 2-Butoxyethanol-Induced Hemolytic Anemia and Studies to Elucidate the Underlying Mechanisms. GHANAYEM, B. I., SANCHEZ, I. M., AND MATTHEWS, H. B. (1992). Toxicol. Appl. Pharmacol. 112, 198-206. Early work demonstrated that a single administration of 2butoxyethanol (BE) causes acute hemolytic anemia in rats. Current studies were undertaken to investigate the effect of repetitive daily dosing of BE on the hematologic parameters of male F344 rats. Treatment of rats with BE daily (125 mg/kg/day) for 1 to 3 consecutive days resulted in a time-dependent increase in the hemolysis of erythrocytes. However, when daily treatment with BE continued beyond 3 days, the number of erythrocytes began to rebound and approached pretreatment levels within 12 days despite continued daily exposure, suggesting development of tolerance to the hemolytic effect of BE. In viva and in vitro studies were designed to investigate the underlying mechanism(s) of tolerance to the hematotoxicity of BE. Rats were treated with 125 mg BE/kg/day for 3 days followed by a 7-day recovery. At the end of this recovery period, rats were challenged with a single 125 or 250 mg BE/kg dose and the hematologic profiles were assessed at 2, 8, and 24 hr later. A significant decline in the sensitivity of BE-pretreated/recovered rats compared to vehiclepretreated rats was observed. Further, in vitro incubation of blood obtained from BE-pretreated/recovered with the hematotoxic metabolite of BE, 2-butoxyacetic acid (BAA), revealed that erythrocytes obtained from these rats were significantly less sensitive to BAA than those obtained from normal rats. These studies suggested that tolerance is due, at least in part, to the lesser sensitivity of young erythrocytes formed during the regeneration process. In another study, rats were rendered anemic by bleeding followed by a 7-day recovery. BE administration to bled/recovered rats demonstrated that these rats were less sensitive than rats which were not subjected to bleeding. In vitro incubation of blood obtained from the bled/recovered animals with BAA demonstrated that erythrocytes were significantly less sensitive to BAA than those obtained from control rats. This further conlirmed that young erythrocytes, formed during the regeneration process, were less sensitive to BAA than older erythrocytes. Current data also suggested that it is unlikely that tolerance is ’ To whom
correspondence
should
2-Butoxyethanol (BE), also known as ethylene glycol monobutyl ether, is a major environmental chemical which is utilized in the manufacturing of a wide range of domestic and industrial products. The 1988 annual U.S. production of BE was estimated at over 400 million pounds (ITC, 1988). Administration of a single dose of BE to laboratory rats causes dose-dependent acute hemolytic anemia which is characterized by a decrease in the number of circulating red blood cells, hemoglobin concentration, and hematocrit (Carpenter et al., 1956; Bartnick et al., 1987; Ghanayem et al., 1987a). Furthermore, BE-induced hemolytic anemia was associated with secondary hemoglobinmia and an increase in the spleen wt/body wt ratio (Ghanayem et al., 1987a). In viva and in vitro studies conducted to elucidate the mechanism(s) of BEinduced effects on erythrocytes demonstrated that hemolysis is preceded by swelling [increased hematocrits (HCT) and mean cell volumes (MCV)] and blood ATP depletion (Ghanayem et al., 1990; Ghanayem, 1989). However, it remains unclear if swelling or ATP depletion is the primary effect of BE. In another study by Grant et al. (1985), BE was administered at 500 or 1000 mg/kg/day for up to 22 consecutive days. BE caused hemolytic anemia, splenic extramedullary hemopioesis, hyperplasia of both spleen and bone marrow, and reticulocytosis. Although we reported that human erythrocytes were significantly less sensitive to 2-butoxyacetic acid (BAA) than rat erythrocytes in vitro (Ghanayem, 1989), another report indicated that hemolysis of erythrocytes occurred in a human who ingested a window cleaning solution containing BE (Rambourg-Scherpens et al., 1988).
he addressed.
0041-008X/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
198
TOLERANCE
TO 2-BUTOXYETHANOL
Work performed in this laboratory and others revealed that BE is metabolized to BAA, BE-glucuronide and BEsulfate conjugates (Ghanayem et al., 1987b), and ethylene glycol (Medinsky et al., 1990). Investigation of the metabolic basis of BE-induced hemolytic anemia in rats showed that metabolic activation of BE to BAA via the alcohol and aldehyde dehydrogenases is a prerequisite for the development of hemolytic anemia and BAA is the proximate hematotoxic metabolite (Ghanayem et al., 1987a; Ghanayem, 1989). The initial objective of the current studies was to investigate the effect of daily dosing of male F344 rats with BE for up to 12 days. These studies showed that daily dosing of rats with BE resulted in tolerance development to BE-induced hemolytic anemia. Subsequently, additional in vivo and in vitro studies were undertaken to investigate the mechanism(s) involved in the development of tolerance to the hemolytic effects of BE. MATERIALS
AND METHODS
Chemicals. BE of >99% purity was purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-( l-[14C] Butoxy)ethanol was purchased from New England Nuclear (Boston, MA). The specific activity of BE was 6.32 mCi/ nmol with greater than 99% radiochemical purity. BAA was synthesized as previously described (Ghanayem ef al., 1989). Animafs and treatments. Male Fischer 344 rats (lo- 14 week) were obtained from Charles River Breeding Laboratories (Raleigh, NC), maintained on NIH 31 diet and water ad libitum, and allowed a minimum of 1 week adjustment to our facilities (12-hr dark/light cycle, 2 l-23°C ambient temperature, and a 40-60% relative humidity) prior to inclusion in the present studies. BE dosing solutions were prepared by mixing with water to obtain a dose volume of 5 ml/kg and administered to rats by gavage. Repeat exposure of rats lo BE. At an age of 10-14 weeks, rats were randomly divided into six groups (six rats/group) and treated by gavage as follows: -Group 1 received 5 ml water/kg body wt (BW) daily for 12 consecutive days (control); -Group 2 received 125 mg BE in 5 ml water/kg BW daily for 12 consecutive days; -Group 3 received 125 mg BE in 5 ml water/kg BW daily for 6 consecutive days; -Group 4 received 125 mg BE in 5 ml water/kg BW daily for 3 consecutive days; -Group 5 received 125 mg BE in 5 ml water/kg BW daily for 2 consecutive days; and -Group 6 received a single dose of 125 mg BE in 5 ml water/kg BW. Twenty-four hours after the last dose, rats were anesthetized in a CO2 chamber and two blood samples were collected from the retroorbital venous plexus. One sample was collected into a 0.75-ml capacity Microvettes KE containing 1.5 mg EDTAK2 as anticoagulant. The second blood sample was collected from the same animals using acetate:citrate:dextrose (ACD) solution as anticoagulant (blood:ACD ratio is 4: 1) to be used for the determination of blood ATP concentration. After blood collection, rats were killed by placement in a COZ chamber and the spleen and liver were removed free of other tissues and weighed. Organ weight/ 100 g body wt ratios were calculated and presented as such. Blood samples were analyzed using an Hl hematology analyzer (Technicon, Tarrytown, NY) and the following pa-
HEMATOTOXICITY
199
rameters were measured: red blood cells (RBC) and white blood cell counts, hemoglobin concentrations (HGB), HCT, MCV, mean cell hemoglobin, mean cell hemoglobin concentration, and platelet counts. In addition, packed cell volumes (PCV) were determined on all blood samples. Only parameters which were affected by BE are presented. Blood ATP concentrations were determined in blood samples containing ACD (anticoagulant) as follows: Packed cell volume was immediately determined and blood samples were mixed with trichloroacetic acid, vortexed, and left in an ice cold water bath for 10 min. Samples were centrifuged at 3000g for 10 min and the supematant was aspirated and used for spectrophotometric ATP determination as previously described (Ghanayem, 1989). Hematotoxicity of BE in BE-pretreated/recovered rats in vivo. Rats (lo14 week) were randomized into two groups. Group A received 125 mg BE/ kg/day for 3 consecutive days by gavage (BE-pretreated/recovered). Group B received water, the vehicle, for the same time period. Animals in both groups were then allowed to recover without treatment for 7 days. Groups A and B are refered to as BE-pretreated/recovered and untreated rats, respectively. BE-pretreated/recovered and untreated rats were divided into three subgroups each and treated as follows: subgroup 1 received 5 ml water/ kg (vehicle); subgroup 2 received 125 mg BE/5 ml water/kg; and subgroup 3 received 250 mg BE/5 ml water/kg. Three to five rats from each of the vehicle, low, and high BE dose subgroups were anesthetized in a CO* chamber at 2. 8, or 24 hr after dosing, and two blood samples were collected from the retroorbital venous plexus. One blood sample was collected using EDTA as an anticoagulant and analyzed using an H 1 hematology analyzer as described above. The second blood sample was collected using ACD as an anticoagulant and utilized for the determination of PCV and ATP concentration in blood. After blood collection, rats were killed by placement in a COZ chamber and the spleen was removed and weighed. The spleen wt/lOO g body wt was calculated and presented as such. Effect ofBAA on bloodfrom BE-pretreated/recovered rats in vitro. Rats were treated with 125 mg BE/kg/day for 3 consecutive days and allowed a 7-day recovery (BE-pretreated/recovered). Blood was collected from CO2 anesthetized rats (>5) by cardiac puncture using ACD as an anticoagulant and pooled. Blood was incubated with BAA at a final concentration of 0, 0.5, 1.0, or 2.0 mM. Samples were incubated at 37°C for 0.5, 1.0, 2.0, or 4.0 hr. At the end of the incubation period, PCV and blood ATP concentrations were measured as described above. Hematotoxicity of BE in bled/recovered rats in vivo. Rats ( IO- I4 week) were lightly anesthetized in a CO2 chamber, bled (4-5 ml blood/rat) via the retroorbital venous plexus, and were allowed a 7day holding period to permit the recovery of the hematopoietic system (bled/recovered rats). Rats were randomized into three groups. Group A received water, the vehicle; group B received a single dose of 125 mg BE/kg BW by gavage; and group C received a single dose of 250 mg BE/kg BW by gavage. Three to four rats from each of the vehicle, low, and high BE dose groups were anesthetized in a CO2 chamber at 2,8, or 24 hr after dosing; a blood sample was collected from the retroorbital venous plexus using EDTA as an anticoagulant and analyzed using an H 1 hematology analyzer as described above. After blood collection, rats were euthanized by placement in a COZ chamber and the spleen was removed and weighed. The spleen wt/lOO g body wt. was calculated and presented as such. Effect of BAA on blood from bled/recovered rats in vitro. Rats were lightly anesthetized in a CO2 chamber and bled (4-5 ml blood/rat) via the retroorbital venous plexus. Animals were allowed a 7-day holding period to allow for the recovery of the hematopoietic system. Using cardiac puncture of CO2 anesthetized animals, blood was collected using ACD as an anticoagulant. Blood was pooled and the effect of BAA on PCV and ATP concentration was investigated as outlined in the previous section. Eflect of multiple dosing of rats with BE on its own disposition and metabolism. Rats were treated with 125 mg/kg BW/day of unlabeled BE for 3 or 7 days by gavage followed by a single dose of 125 mg/kg BW of r4CBE (40-50 &i/kg) on Days 4 and 8. respectively. Another untreated three
200
GHANAYEM,
0 (control) 1 2 3 6 12
AND MATTHEWS
TABLE 1 Parameters” of Male F344 Rats Treated with 2-Butoxyethanolb Daily for Various Periods of Time
Hematologic Days of treatment
SANCHEZ,
RBC (106/d) 8.9 5.1 4.2 4.4 5.9 6.8
+ * + f + f
HGB Wl)
0.2 0.2* 0.4* 0.2* 0.1* 0.1*
15.3 iz 0.2 8.7 5 0.2* 7.2 f 0.6* 8.0 f 0.3* 11.2 f 0.2* 13.0 + 0.1*
HCT 6) 46.8 28.4 24.0 26.1 38.5 43.0
f + + -t + +
MCV (fl)
0.8 0.9* 1.8* 0.9* 0.7* 1.0:
52.5 55.5 56.9 59.5 65.5 63.3
k * + f f f
ATP (rc.mol/g HGB)
0.2 0.5* 0.9* 0.5* 1.6* 0.5*
4.1 4.8 5.9 7.0 7.4 5.3
Recticulocytes (million/~l)
+ 1.0 k 1.2; f 1.9* iz 4.6* rf: 1.7* + 0.8*
0.14 0.23 0.26 0.36 0.87 0.36
k + + f f f
0.05 0.06* 0.03* 0.02* 0.14* 0.06*
a Data are presented as the mean _+ SE of four to five rats. * 2-Butoxyethanol was administered daily at 125 mg/kg/day and rats were killed 24 hr after the last dose. * Significantly different from control at d< 0.05. - -’ -
rats received the same dose of 14C-BE. Rats treated with radiolabeled BE were immediately placed in glass metabolism cages which allowed separate collection of fecesand urine. Expired volatiles and 14C02were also collected. Disposition and urine analysis of BE metabolites were performed as previously described (Ghanayem et al., 1987b). Statisticul analysis. Statistical analysis was performed using a pairwise comparison of variance (pooled t test). Values were considered statistically significant at p < 0.05.
RESULTS Efect of repeat exposure of rats to BE. As observed in earlier studies (Ghanayem et al., 1990) administration of a single dose of BE (125 mg/kg) to rats resulted in significant hemolysis of erythrocytes as evidenced by a decreased RBC, HGB, and HCT (Table 1). This effect became more pronounced following the third day of dosing, but gradual recovery was observed thereafter. With continued repeated exposure to BE beyond Day 3, rats exhibited a gradual increase in RBC and HGB and approached pretreatment levels by Day 12 (Table 1). HCT followed a similar pattern as RBCs and HGB as dosing continued. On the other hand, MCV increased as the dosing regimen was extended to 6 days and declined to remain above control levels throughout the 12day study (Table 1). ATP concentration, MCV, and the number of reticulocytes increased after 6 days and then declined by Day 12 (Table 1). Reticulocytosis and increased ATP concentration were evident after the second day of dosing and persisted in BE-treated rats throughout the study. The effect of repeat dosing of rats with BE on the weight of the spleen and liver is shown in Fig. 1. Spleen wt/body wt ratios markedly increased to a maximum after 6 days from which they declined between Days 6 and 12 (Fig. 1). Liver wt/body wt ratios were minimally affected by repeat dosing and, following a moderate decline on Days 3 and 6, they were increased on Day 12 compared to those observed in controls (Fig. 1). Hematologic parameters of BE-pretreated/recovered rats treated with BE in vivo. Comparison of BE-induced he-
matotoxicity in BE-pretreated/recovered and untreated rats revealed that BE-pretreated/recovered rats were significantly less sensitive to the hemolytic effects of BE than untreated rats (rats which were not previously exposed to BE; Table 2). The RBC and HGB declined in a dose- and time-dependent manner, however, there was a minimal decline in these parameters in rats pretreated with the low dose of BE (Table 2). HCT increased at 2 hr and remained near or slightly below control in BE-pretreated/recovered rats. In contrast, rats which were not previously exposed to BE exhibited an increase in HCT early after treatment which later declined in a dose- and time-dependent manner (Table 2). Treatment
160
I \‘\ ‘\ ,I ‘\ ‘\ ‘\
0
2
4
6
6
10
‘\ \ I
12
14
TIME (days)
FIG. 1. Effect of administration of BE (125 mg/kg/day) on the liver (-) and spleen (---) weights/body wt ratios of F344 male rats. Values are presented as percentage control and are the mean + SE of three to six rats.
TOLERANCE
TO 2-BUTOXYETHANOL
201
HEMATOTOXICITY
TABLE 2 Comparison of the Effects of 2-Butoxyethanol” on the Hematologic Parameters of Untreated and BE-Pretreated/Recovered” Male F344 Rats Time (hr)
RBC’
HGB’
HCTC
MCV’
Untreated ( 125 mgfkg)
2 8 24
87.6 zk 1.6 82.9 t 3.9 66.4 f 2.9
88.0 2 1.5 85.4 i- 2.9 65.4 + 2.8
114.3 rt 1.7 92.9 * 2.6 61.3 + 3.8
116.4 + 2.0 101.1 z!z2.3 91.0 + 0.3
59.0 f 2.9 68.3 f 1.5 10.5 f 3.5
0.13 + 0.03 0.21 k 0.08 0.22 * 0.02
BE-Pretreated (125mgk)
2 8 24
95.6 f 0.6 94.6 f 1.3 98.9 -c 0.7
94.8 f 0.2 93.7 f 1.3 98.6 -c I.0
112.6 k 0.6 101.5 z!z0.8 102.3 EL 1.5
117.5 + 0.6 107.3 f 0.6 102.8 -c 1.5
116.1 f 1.4 93.4 f 1.2 105.4 iz 5.1
0.44 + 0.05 0.32 + 0.06 0.50 f 0.07
Untreated (250w/kg)
2 8 24
79.7 f 1.3 58.8 f 1.6 56.1 + 0.7
81.5 + 1.4 85.4 f 2.9 55.5 f 0.7
108.8 + 1.61 13.1 * 2.2 61.4 k 1.5
122.0 + 0.0 111.9?2.9 98.8 + 2.8
39.3 + 2.9 56.9 rf: 2.6 64.9 + 1.7
0.17 + 0.03 0.30 * 0.05 0.30 k 0.02
BE-Pretreated (250 w/W
2 8 24
92.6 + 1.1 91.2 + 1.4 90.6 f 1.1
90.2 + 0.8 91.4 r 0.8 91.6 + 1.8
113.9 + 1.4 106.8 t 2.2 95.00 f 1.2
122.6 + 0.56 118.1 r 2.0 104.5 * 1.5
76.1 zk 3.9 85.3 i 1.5 92.3 ?I 2.4
0.41 + 0.04 0.48 k 0.02 0.50 * 0.03
Treatment
ATP
Recticulocytesd
a 2-Butoxyethanol was administered by gavage at a dose volume of 5 ml/kg. b Rats were treated with 125 mg BE/kg for 3 consecutive days and allowed 7 days recovery. ’ Data are presented as percentage of the corresponding control and are the mean f SE of four to five rats. d Reticulocvtes are presented in millions/u1 of blood. Reticulocvtes in untreated and BE-pretreated controls were 0.13-0.23 and 0.42-0.45 million/r] of blood, respectively.-
of BE-pretreated/recovered rats with the high BE dose (250 mg/kg) caused more pronounced hematotoxicity than the low dose ( 125 mg BE/kg); however, this effect remained significantly less than that observed in untreated rats receiving the same BE dose (Table 2). BE-pretreated/recovered rats were also less sensitive to the increase in MCV and ATP depletion caused by BE than rats which were not previously exposed to BE (Table 2). In addition, the number of reticulocytes was significantly higher in rats pretreated with BE than in untreated controls (Table 2). Comparison of the spleen w/body wt ratio in these rats is shown in Fig. 2 and demonstrated that the increase caused by BE is significantly less in BE-pretreated rats than in untreated rats (Fig. 2). Efect of BAA on blood from BE-pretreated/recovered rats in vitro. Incubation of blood obtained from rats pretreated with BE for 3 consecutive days and allowed a 7-day recovery with the toxic metabolite of BE, BAA, suggested that RBCs are significantly less sensitive to BAA than those obtained from untreated rats (Figs. 3 and 4). Figure 3 shows that the increase in PCV caused by BAA in blood from untreated rats was significantly greater than that caused in blood from BE-pretreated rats. Similarly, the effect of BAA on ATP concentration in blood from both the BE-pretreated/recovered and untreated rats revealed that pretreatment of rats with BE decreased the susceptibility of erythrocytes to the effect of BAA in vitro (Fig. 4). Both effects of BAA became more dramatic as the concentration of BAA and the incubation time increased.
Hematologic parameters of bled/recovered rats treated with BE in vivo. Comparison of the hematotoxicity of BE in bled/recovered and controls (not bled) rats revealed that bled/
0
5
10
15
20
25
TIME (hr)
FIG. 2. Effect of administration of a single 250 (0) and 125 (m) mg BE/ kg dose on the spleen wt/body wt ratio of control (-) and BE-pretreated/ recovered (---) male F344 rats. Values are presented as percentage control and are the mean f SE of three to six rats.
202
GHANAYEM,
SANCHEZ, AND MATTHEWS
160-
g 3
140-
g= -lg d,’ 00 09 ue :: d
130.
120-
110-
0
1.0
2.0
3.0 TIME
4.0
(hr)
FIG. 3. Effect of BAA concentration and time of incubation on packed cell volume of blood obtained from control (-) and BE-pretreated/recovered (---) male F344 rats. Values are presented as percentage control and are the mean + SE of six to nine determinations. BAA final concentrations in blood were: A, 2.0 mM: 0, 1.0 mM; n , 0.5 mM.
recovered rats were significantly less sensitive to the hemolytic effects of BE than controls (Table 3). The RBCs and HGB declined in a dose- and time-dependent manner; however, the decline in bled/recovered rats treated with the 125 mg BE/kg BW was relatively minimal (Table 3). HCT increased at 2 hr and remained near pretreatment levels in bled/recovered rats. In contrast, BE administration to controls resulted in an early increase in HCT and then declined in a dose- and time-dependent manner (Table 3). Although the number of reticulocytes in bled/recovered rats prior to exposure to BE was significantly higher than in the corresponding controls, this trend increased or remained the same after exposure to BE (Table 3). Treatment of bled/recovered rats with the high BE dose (250 mg/kg) caused more pronounced hematotoxicity than the low dose (125 mg/kg); however, these effects continued to be less pronounced than those observed in control rats receiving the same BE dose (Table 3). Comparison of the spleen wt/body wt ratio demonstrated that there was a significantly greater increase caused by BE in control rats compared to bled/recovered rats (data not shown). Efsect of BAA on blood from bled/recovered rats in vitro. Comparison of the effect of BAA on blood from bled/ recovered and normal rats is shown in Figs. 5 and 6. Blood obtained from bled/recovered rats was less sensitive to BAA-
induced increases in PCV and ATP depletion. These effects were more pronounced at higher BAA concentrations and at later incubation times (Figs. 5 and 6). Eflect of multiple dosing of rats with BE on its own disposition and metabolism. The effect of multiple dosing of rats with BE on its own metabolism and disposition is shown in Tables 4 and 5. CO2 exhalation and urinary excretion were shown as the two major routes of BE elimination (Ghanayem et al., 1987b). The percentage of the administered BE dose eliminated as CO1 and in the urine remained unaffected by pretreatment of rats with BE for 4 or 8 days. Seventeen to 20% of a 125 mg BE/kg dose was exhaled as 14C02 by rats receiving 1, 4, or 8 doses of BE. Urinary excretion of BE-derived radioactivity also remained unchanged and ranged from 55 to 65% of the administered dose in 24 hr (Table 4). Results of the HPLC analysis of the urinary metabolites of BE in rats treated with BE for 1, 4, or 8 days are shown in Table 5. No significant difference in the percentage of dose excreted in the urine as BAA (hematotoxic metabolite) was detected between the three groups of rats (Table 5). Although the percentage of dose excreted as BAA in the urine at 8-24 vs O-8 hr has increased within each of the groups, it remained essentially the same in the three groups. In addition, no major differences in the percentage of dose excreted as BE and BE-glucuronide and BE-sulfate
1
30&Y, 0 1.0
I 2.0
I 3.0
4.0
TIME (hr)
FIG. 4. Effect of BAA concentration and time of incubation on ATP concentration in biood obtained from control (-) and BE-pretreated/recovered (---) male F344 rats. Values are presented as percentage control and are the mean f SE of six to nine determinations. BAA concentrations in blood were: A, 2.0 mM; 0, I.0 mM: n , 0.5 mM.
TOLERANCE
TO 2-BUTOXYETHANOL
203
HEMATOTOXICITY
TABLE 3
Comparison of the Effects of 2-Butoxyethanol” on the Hematologic of Control and PrebledlRecovered* Male F344 Rats
Parameters
Time U-4
RBC’
HGB’
HCT’
Control ( 125 mg BE/kg)
2 8 24
88 f 1.6 83 k 3.9 66 k 2.9
88 f 1.5 85 f 2.9 65 f 2.8
114 f 1.7 93 + 2.6 67 k 3.8
116 + 2.0 101 f 2.3 91 f 0.3
0.13 rt 0.03 0.21 + 0.08 0.22 Ik 0.02
Prebled ( 125 mg BE/kg)
2 8 24
94 f 0.9 91 f 3.8 88 f 0.6
91 + 0.6 94 + 2.5 92 + 0.5
115kO.6 105 + 2.5 94 f 0.8
121 + 1.0 116 k 1.1 106 f 1.0
0.54 + 0.0 1 0.66 + 0.04 0.51 * 0.02
Control (250 mg BE/kg)
2 8 24
80 + 1.3 59 + 1.6 56 f 0.7
82 + 1.4 85 + 2.9 56 + 0.7
109 f 1.61 73 f 2.2 61 rl 1.5
122 + 0.0 112 ?I 2.9 99 f 2.8
0.17 f 0.03 0.30 f 0.05 0.30 * 0.02
Prebled (250 mg BE/kg)
2 8 24
88 + 1.5 83 + 2.2 82 + 2.0
87 + 1.5 86 f 1.7 83 f 2.4
114 + 1.5 101 f 4.0 91 f 2.7
130 + 1.7 121 + 3.0 111 + 1.5
0.47 + 0.02 0.63 + 0.06 0.52 + 0.01
Treatment
MCV’
Recticulocytesd
” 2-Butoxyethanol was administered by gavage at a dose volume of 5 ml/kg. b Rats were bled and allowed a ‘I-day recovery prior to BE administration. ’ Data are presented as percentage of the corresponding control and are the mean + SE of three to five rats. d Reticulocytes are presented in million/r1 blood. Reticulocytes in control and bled/recovered rats were 0.13-0.23 and 0.39-0.56 million/r1 blood, respectively.
conjugates were observed in rats treated with BE for 1,4, or 8 days (Table 5). DISCUSSION
Administration of a single dose of BE to laboratory animals caused dose- and time-dependent hemolytic anemia (Carpenter et al., 1956; Ghanayem et al., 1987a). BE-induced hemolytic anemia was characterized by an early swelling of erythrocytes (increased HCT and MCV) associated with ATP depletion, resulting in hemolysis and decline in the number of circulating erythrocytes, hemoglobin concentration, and hematocrit (Ghanayem, 1989; Ghanayem et al., 1990). In addition, BE caused secondary hemoglobinurea and increased spleen wt/body wt ratio (Ghanayem et al., 1987a). Spleen enlargement was attributed to sequestration of swelled/deformed RBCs and was directly related to the effect of BE on RBCs. Current studies were undertaken to investigate the effects of repeat exposure of male F344 rats to a relatively low dose of BE. Results presented in this report indicated that repetitive daily dosing of rats with 125 mg BE/kg/day resulted in the development of hemolytic anemia, which reached a maximum after 2-3 days. Continued administration of the same BE dose beyond the third day resulted in gradual recovery from the anemic condition in association with tolerance development to the hemolytic effect of BE. This tolerance was characterized by a gradual recovery of the hematologic pa-
rameters (increase in RBCs, HGB, HCT, and MCV) despite continued daily exposure to BE. In addition, ATP concentration in blood increased with time to reach a maximum after 6 days of dosing. Reticulocytosis (increased number of reticulocytes in blood) was also evident after the second dose of BE and persisted throughout the 12 days of repeated exposure. Both the number of reticulocytes and the ATP concentration in the blood declined after 12 days compared to that observed after the sixth day (Table 1). Although it was not specifically identified, tolerance to BE-induced hemolytic anemia was also detectable upon close examination of other prechronic studies (Krasavage, 1986; Grant et al., 1985). In these studies, administration of BE at doses as high as 1000 mg/kg/day for 6 weeks resulted in significantly less pronounced hemolytic anemia than we observed 24 hr after a single 250 or 500 mg BE/kg dose (Ghanayem et al., 1990). It is likely that tolerance was not specifically identified in early studies (Krasavage, 1986; Grant et al., 1985) because ~~~mparison of preclironic and acute (the effect of a single BE dose was not included in the studies) effects of BE were not included. In previous studies, we have demonstrated that metabolic activation of BE to BAA via the alcohol and aldehyde dehydrogenases is a prerequisite for hemolysis of erythrocytes in rats receiving BE and that BAA is the proximate hemolytic agent (Ghanayem et al., 1987a). Other metabolites of BE appear to have little effect on RBCs. Therefore, one hypothesis to explain the biological basis of tolerance development
204
GHANAYEM,
SANCHEZ, AND MATTHEWS
160-
1001’ 0
1.0
2.0
3.0
TIME
0
4.0
1.0
has initially centered on the premise that continued administration of BE may have caused a gradual inhibition of BE metabolism to BAA or induction of an alternative metabolic pathway(s). To address this hypothesis, we examined the metabolism of 14C-labeled BE by rats pretreated with unlabeled BE for 4 or 8 consecutive days. These studies revealed that no quantitative or qualitative alterations of BE metabolism and disposition were caused by repeat exposure to BE compared to those observed in rats treated with a single dose. Elimination of 14C02 and BE-derived radioactivity in the urine of rats receiving multiple doses of BE was essentially the same as in rats receiving a single BE dose (Table 4). In addition, the ratios of BAA (hematotoxic metabolite), the glucuronide and sulfate conjugates, and parent BE excreted in the urine of rats treated for 4 or 8 days were relatively similar to those from rats treated with a single BE dose (Table 5). Therefore, it is unlikely that tolerance development to the hemolytic effects of BE is caused by increased detoxification of BE or inhibition of BE metabolism to BAA. It is well known that aging of erythrocytes results in several biochemical and biophysical changes. For example, the concentrations of glutathione, superoxide dismutase, ATP, and 2,3diphosphoglycerate decrease while the concentration of lipid peroxides increases during the aging of erythrocytes (Abraham et al., 1978; Glass and Gershon, 1981; Turner et
1 3.0
TIME
(hr)
FIG. 5. Effect of BAA concentration and time of incubation on packed cell volume of blood obtained from control (-) and previously bled/recovered (---) male F344 rats. Values are presented as percentage control and are the mean + SE of six to nine determinations. BAA concentrations in blood were: A, 2.0 mM; 0, 1.0 mM; n , 0.5 mM.
1 2.0
1 4.0
1
(hr)
FIG. 6. Effect of BAA concentration and time of incubation on ATP concentration in blood obtained from control (-) and previously bled/ recovered (---) male F344 rats. Values are presented as percentage control and are the mean f SE of six to nine determinations. BAA concentrations in blood were: A, 2.0 mM; 0, 1.0 mM; n , 0.5 mM.
al., 1974; Shiga et al., 1979; Glass and Gershon, 1984). In addition, erythrocytes become more osmotically fragile and less deformable during the aging process (Shiga et al., 1979). These data suggest that erythrocytes undergo significant changes during aging which may result in increasing their susceptibility to chemical-induced hemolysis. We therefore hypothesized that there is a direct relationship between the age of erythrocytes and their sensitivity to the hemolytic effect of BE with older erythrocytes being more susceptible. Early in the dosing regimen with BE, the highly sensitive old erythrocytes undergo hemolysis leaving the less sensitive young erythrocytes. The selective hemolysis of older erythrocytes
TABLE 4 Effect of Dosing Rats with BE” for up to 8 Days on Its Own Metabolism
‘TO2 Urine
One dose
Four doses
Eight doses
17.2 + 0.5b
19.8 k 1.3 57.3 f 5.4
20.0 * 1.4 55.1 + 4.0
64.6 f 5.7
0 Unlabeled BE was administered for 3 or 7 days followed by ‘%-BE on Day 4 (four doses) and Day 8 (eight doses). A third group received a single 14C-BE (one dose). b Values are the mean f SE of three animals.
TOLERANCE
TO 2-BUTOXYETHANOL
205
HEMATOTOXICITY
TABLE 5 Comparison of the Effect of Daily Dosing of Rats with BE” for 1,4, or 8 Days on the Excretion of BE Urinary Metabolites Four doses
One dose Metabolitesb BAA BEG BES BE
8 hr 67.8 24.0 3.5 2.2
+- 3.0’ -t 2.0 I? 0.3 + 1.0
24 hr 91.6 k 1.1 6.6 t 1.1 ND ND
24 hr
8 hr 68.5 11.9 5.1 6.8
f + + f
Eight doses
0.7 1.4 0.2 1.1
8 hr
84.2 f 4.8 8.1 f 0.9 ND ND
65.5 24.2 2.4 4.9
+ f + f
24 hr 5.7 5.4 0.3 3.6
90.2 ?I 0.4 5.2 F 1.4 ND ND
a Unlabeled BE was administered for 3 or 7 days followed by 14C-BE on Day 4 (four doses) and day 8 (eight doses). A third group received a single 14CBE (one dose). b BAA, 2-butoxyacetic acid, BEG, glucuronide conjugate of BE; BES, sulfate conjugate of BE. ’ Values are the mean -CSE of three animals.
in association with the development of reticulocytosis during the process of active blood regeneration may lead to tolerance to the hemolytic effects of BE. This hypothesis was addressed by investigating the effect of BE and BAA on erythrocytes of anemic/recovered rats in vivo and in vitro, respectively. Anemia was induced either by treatment of rats with BE (3 consecutive days at 125 mg/kg/day) or by bleeding. These animals were then allowed to recover for 7 days. Results presented in this report demonstrated that anemic/recovered animals were significantly less sensitive to BE than control rats. However, tolerance to BE was more pronounced in BEpretreated/recovered rats than in bled/recovered rats. This discrepancy is very likely related to the fact that bleeding resulted in the loss of RBCs of ah ages while BE-pretreatment resulted in the selective removal (hemolysis) of the sensitive older cells. Furthermore, erythrocytes obtained from anemic/ recovered rats were found to be less sensitive to BAA in vitro than erythrocytes obtained from control rats. These data, especially the effect of BAA in vitro, confirm the conclusion that tolerance to BE is not mediated by changes in BE metabolism and is most likely caused by the increased susceptibility of erythrocytes to BE/BAA as a function of age. Tolerance development to hemolytic agents such as the antimalarial drug primaquine has long been recognized and documented and was thought to be caused by the higher susceptibility of older RBCs vs younger cells (Kellermeyer et al., 1962). Although the mechanism of RBCs hemolysis by BE is not fully understood, it is unlikely that the cellular basis for tolerance development to primaquine and BE is identical. There is evidence which suggests that the pattern and mechanisms of hemolysis caused by both agents are different. For example, BE-induced hemolysis is preceded by swelling of erythrocytes while primaquine-induced hemolysis is not mediated by erythrocytic swelling. In conclusion, tolerance to BE-induced hemolysis developed upon repeated daily exposure of animals to this chemical. The mechanism(s) underlying this phenomena may be
due, at least in part, to the selective hemolysis of older erythrocytes and their replacement during hematopoiesis with less sensitive young cells. However, the specific change(s) in aged erythrocytes which may increase their sensitivity to hemolysis by BE or BAA remain to be investigated. This report further suggests that chronic administration of BE may cause anemia; however, this effect is unlikely to be as severe as that caused by a single dose of BE. Chronic administration is expected to result in severe anemia early on followed by gradual development of tolerance and equilibrium between hematopoiesis and hemolysis of erythrocytes. This equilibrium is expected to be associated with a decrease in the halflife of erythrocytes leading to lower number of erythrocytes in rats treated with BE chronically compared to controls. It is, therefore, expected that chronic administration of BE may cause hematopoiesis to be sustained above the normal rate, which may subject the hematopoietic system to sustained stress with possible adverse consequences on systems such as spleen and bone marrow. ACKNOWLEDGMENT The authors thank Ms. Sandra Ward for her excellent technical help.
REFERENCES Abraham, E. C., Taylor, F. J., and Lang, C. A. (1978). Influence of mouse age and erythrocyte age on glutathione metabolism. Biochem. J. 174, 819-825. Bartnik, F. G., Reddy, A. K., Klecak, G., Zimmermann, V., Hostynek, J. J., and Kunstler, K. (1987). Percutaneous absorption, metabolism, and hemolytic activity of n-butoxyethanol. Fundam. Appl. Toxicol. 8,59-70. Carpenter, P. C., Pozzani, C. U., Weil, C. S., Nair III, H. J., Keck, G. A., and Smyth, H. F. (1956). The toxicity of butyl cellosolve solvent. Arch. Ind. Health
14, 114-131.
Ghanayem, B. I. (1989). Investigation of the mechanisms of 2-butoxyethanol induced hemolytic anemia in rats and assessment of human risk in vitro. Biochem. Pharmacol. 38, 1679-1684. Ghanayem, B. I., Burka, L. T., and Matthews, H. B. (1987a). Metabolic basis of ethylene glycol monobutyl ether (2-butoxyethanol) toxicity: Role
206
GHANAYEM,
SANCHEZ,
of alcohol and aldehyde dehydrogenases. J. Pharmacol. Exp. Ther. 242, 222-231. Ghanayem, B. I., Burka, L. T., Sanders, J. M., and Matthews, H. B. (1987b). Metabolism and disposition of ethylene glycol monobutyl ether (2-butoxyethanol) in rats. Drug Metab. Dispos. 15, 478-484. Ghanayem, B. I., Burka, L. T., and Matthews, H. B. (1989). Structureactivity relationships for the in vitro hematotoxicity of n-alkoxyacetic acids, the toxic metabolites ofglycol ethers. Chem. Biol. Interact. 70,339352. Ghanayem, B. I., Ward, S. M., Blair, P. C., and Matthews, H. B. (1990). Comparison of the hematologic effectsof 2-butoxyethanol using two types of hematology analyzers. Toxicol. Appl. Pharmacol. 106, 341-345. Glass, G. A., and Gemhon, D. (198 1). Enzymatic changes in rat erythrocytes with increasing cell and donor age: Loss of superoxide dismutase activity associatedwith increases in catalytically defective forms. Biochem. Biophys. Res. Commun. 103, 1245-1253. Glass, G. A., and Gershon, D. (1984). Decreased enzymatic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell and donor aging. Biochem J. 218,531-537. Grant, D., Slush, S., Jones, B. W., Gangolli, D. S., and Butler, W. H. (1985). Acute toxicity and recovery in the hematopoietic system of rats after
AND MATTHEWS
treatment with ethylene glycol monomethyl and monobutyl ethers. Tox77, 187-200. ITC/International Trade Commission Report on Annual Production of the Year 1987. September 1988. Kellermeyer, R. W., Tarlov, A. R., Brewer, G. J., Carson, P. E., and Alving, A. S. ( 1962). Hemolytic effect of therapeutic drugs: Clinical considerations of the primaquine-type hemolysis. JAMA 180, 388-394. Krasavage, W. J. (1986). Subchronic oral toxicity of ethylene glycol monobutyl ether in male rats. Fundam. Appt. PharmacoL 6, 349-355. Medinsky, M. A., Singh, G., Bechtold, W. E., Bond, J. A., Sabourin, P. J., Birnbaum, L. S., and Henderson, R. F. (1990). Disposition of three glycol ethers administered in drinking water to male F344/N rats. Toxicol. Appl. icol. Appl. Pharmacol.
Pharmacol.
102,443-445.
Rambourg-Schepens, M. O., Buffet, M., Bertault, R., Jaussaud, M., Joume, B., Fay, R., and Lamiable, D. (1988). Severe ethylene glycol butyl ether poisoning: Kinetics and metabolism pattern. Human Toxicol. 7, 187189. Shiga, T., Maeda, N., Suds, T., Kon, K., and Sekiya, M. (1979). The decreased membrane fluidity of in vivo aged human erythrocytes. Biochem. Biophys. Acta 553, 84-95.
Turner, B. M., Fisher, A. R., and Harris, H. (1974). The age related loss of activity of four enzymes in the human erythrocytes. C/in. Chim. Acta 50, 85-95.
