Toxwology, 72 (1992) 41-50 Elsewer Scientific Pubhshers Ireland Ltd
41
Effects of dimethylformamide on hepatic microsomal monooxygenase system and glutathione metabolism in rats Kazuhiko Imazu a'b, Kazuya Fujishiro b and Naohide Inoue b aThtrd Department of Internal Medwme, School of Medtcme and bDepartment of Envtronmental Toxwology, lnstttute of lndustrtal Ecologwal Scwnce, Umverstty of Occupatzonal and Envtronmental Health 1-1 lsetgaoka Yahatantshtku, Kttalyushu 807 (Japan) (Recewed August 23rd, 1991, accepted December 8th, 1991)
Summary The effects of repeated exposure to N,N-&methylformamlde (DMF) on hepatic mlcrosomal monoox~genase system and glutathlone metabohsm were investigated D M F was administered to W~star male rats by subcutaneous (s c ) rejection at 0 5 ml/kg body weight dally for I week Macroscoplcally, mdd hver swelhng was observed and hver weights slgmficantly increased after 1 week of exposure to DMF Hematologxcal changes were not detected In exposed rats, glutamlc oxaloacetlc transammase, glutamlc pyruvlc transammase, chohnesterase and total cholesterol s~gmficantly increased Hepatic m~crosomal cytochrome P-450 and protoheme decreased by 34% and 24%, respectwely, while mlcrosomal protein and cytochrome b 5 were not affected NADH-ferrlcyamde reductase actwlty decreased by 24% whde NADPH-cytochrome c reductase actwlty showed no change Glutathlone reductase (GR) actwlty showed a slgmficant decrease after the first rejection and remamed depressed throughout the study, with no change m glutath~one peroxldase (GPx) actwlty Glutath~one S-transferase (GST) actwlty showed a slgmficant mcrease at 3 days after D M F treatment and gradually increased by 66% at 1 week In a subsequent experiment with a single administration of D M F (4 ml/kg), reduced glutathlone (GSH) m the hver was decreased by 28"/0 at 8 h, but recovered to control levels by 24 h These results indicate that DMF alters the hepatic mlcrosomal monooxygenase system and glutathlone metabohsm These findings may greatly contribute to the elucldaUon of the pathogenesls of DMF hepatotox~c~ty
Key words Dimethylformamlde, Liver, Cytochrome P-450, Glutathlone, Glutathlone S-transferase
Introduction N,N-dimethylformamide (DMF) is widely used as an organic solvent m laboratories and the chemical industry (e.g. in the manufacture of polymers such as polyvmylchlorlde, polyacrylonltrile and polyurethanes) [1]. It is absorbed through the lungs and very readily through the skin [2]. The primary target organ of D M F toxicity in experimental animals and humans is the liver [3,4]. Occupational exCorrespondence to Kazuhlko Imazu, Third Department of Internal Me&cme, School of Medicine. UOEH, 1-1 Iselgaoka Yahatamsh~ku, K~takyushu 807, Japan 0300-483X/92/$05.00 © 1992 Elsevier Scientific Pubhshers Ireland Ltd Printed and Pubhshed m Ireland
42 posure to D M F causes nausea, appetite loss, abdominal pare, alcohol intolerance and hepatic damage [4-7]. Very recently, Redlich et al [8] reported an outbreak of toxic liver disease among workers exposed to DMF. Little is known of its mechanism of hepatotoxicity. N-acetyl-S-(N-alkylcarbamoyl)cystelnes (AMCC) has been ldentiffed unequivocally in the urine after exposure to D M F in humans and rodents [9] This supports the possibility that conjugation with G S H is one metabolic pathway of D M F in addition to the classical metabolic pathway that D M F receives hydroxylation of one methyl group to yield N-hydroxymethyl-N-methylformamlde ( H M M F ) and N-demethylatlon to yield N-methylformamlde (NMF) and formamlde by cytochrome P-450. However, no detailed studies have been conducted on the effects of D M F on hepatic glutathlone metabolizing enzymes and mxcrosomal monooxygenase system. In this study, we investigated the effects of D M F on hepatic drug metabolizing enzymes, particularly the microsomal monooxygenase system, cytochrome P-450 and glutathione metabolism. Materials and methods
Ammals and chemicals Wistar male rats, approximately 7 weeks of age, were obtained from Selwa Experimental Animals Ltd. (Fukuoka, Japan) They were randomly divided into control and DMF-treated groups and were acclimated to the animal facility for 1 week prior to the experiment. The environmental conditions were a temperature of 24 ± I°C, relative humidity of 55 ± 5°C and a 12 h light-dark cycle (light from 07:00 to 19:00 h). The rats of the exposed group had free access to food (Japan CLEA, CE-2), while the control groups were given the same volume of food that the exposed groups took in the day before (pair-fed controls) Water was available ad hbitum. D M F (98% pure) was purchased from Wako Pure Chemical Industries L t d , Japan. All chemicals used were of reagent grade.
Expertmental destgn D M F was administered by subcutaneous (s.c.) xnlectlon at 0.5 ml/kg body wt (475 mg/kg) daily for 1 week. Control rats were treated with an equal volume of 0 9'/,, NaC1. Eight animals were used for each group Food was removed 24 h before killing. The animals were killed m pairs, one from each group, under deep ether anesthesia about 24 h after the last treatment. Blood was obtained from the subclavian artery with ethylenedlaminetetraacetlc acid, dipotasslum salt (EDTA-2K), as an anticoagulant for blood cell counts, or with heparin sodium salt for other assays. The livers were perfused in situ with cold 0.9% NaC1 and then liver and spleen were removed and weighed. A portion of the hver was homogenized with 3 vol 1.15% KC1 using a Potter-Elvejem homogenizer with a Teflon pestle. To an aliquot of the homogenate was added 1.0 ml metaphosphorlc acid, followed by centrlfugatlon at 10 0000 × g for 30 min in a Beckman L8-M ultracentrifuge The supernatant was used to determine reduced glutathaone (GSH) and oxidized glutathlone (GSSG) content. The rest of the homogenate was centrifuged at 10 000 × g for 20 min and the supernatant centrifuged at 105 000 × g for 60 man in a Beckman L 8 - 5 0 M / E ultracentrifuge. This supernatant, the cytosolic fraction, was used to determine
43 glutathione metabohzlng enzyme activities. The pellet containing the mlcrosomal fraction was used to determine cytochrome P-450, b5 and protoheme content. N A D P H - c y t o c h r o m e c reductase and NADH-ferrlcyanide reductase activity were also measured using the macrosomal fraction G S H is present an high concentrations in most living cells [10] It Is said that the depletion of G S H may be of short duration, since G S H synthesis increases rapidly after acute depletion [11]. So, there is the posslbihty that G S H content in the liver may resume the control level at 24 h after D M F injection. The second study was conducted to confirm the decrease of G S H in the liver. Exposed animals received 4 ml/kg (3800 mg/kg) D M F by s c route, and were killed at specified times (2, 4, 6, 8, 12 and 24 h) following treatment. The control group recexved 0 9°/,, NaCI, followlng the same dosing schedule. Food was removed after D M F injection. G S H content in each laver was determined as described above
Hematological and blochemtcal exammatton A Towa Counter Analyzer (Model E-3000) was used for white blood cell counts (WBC), red blood cell counts (RBC) and determlnatlon of hemoglobin concentration (Hb) and hematocrit (Ht) An Hitachi Counter Analyzer (Model H-710, 736) was used to determine total protein (TP), albumin (Alb), total blhrubin (T-Bll), glutamic oxaloacetic transamlnase (GOT), glutamlc pyruvic transammase (GPT), alkaline phosphatase (Alp), lactate dehydrogenase (LDH), chohnesterase (Ch-E), total cholesterol (T-cho) and triglycerlde (TG) Assay methods The concentration of cytochrome P-450 in laver mlcrosomes was determined from the reduced CO difference spectrum using an extinction coefficient of 91 m M -J cm -1 as reported by Omura and Sato [12]. Cytochrome b5 and protoheme content in the liver was estimated by the methods of Stnttmatter and Vehck [13] and Omura and Sato [12], respectively. N A D P H - c y t o c h r o m e c reductase and N A D H ferracyanide reductase activity were determined by the method of Philhps and Langdon [14]. Glutathione reductase (GR) activity was measured by the method of Carlberg and Mannervak [15] with mo&ficatlon. The reaction mixture contained 1.99 ml of 0.2 M potassium phosphate buffer, pH 7.0, with 2 mM E D T A and 10 ~1 of cytosol. A total of 200/A of GSSG was added to the mixture followed by incubation at 30°C for 10 min. The reaction was initiated an a cuvette by the addition of 200 tA of 2 m M reduced nlCotlnamlde adenine dinucleotlde phosphate ( N A D P H ) in 10 mM Tris-HC1 buffer, pH 7.0. Decrease In absorbance was momtored at 340 nm and 30°C Glutathione peroxidase (GPx) activity was measured by a slightly modified method of Beutler et al [16] The reaction mixture contained 200 tA of 1 M Tris-HC1 buffer, p H 8.0, 40/zl of 0.1 M GSH, 100/xl of 10 IU/ml GR, 100/zl of 1 M N a N 3, 5 #1 of cytosol and 1 vol. of water to bring the final volume to 1.78 ml. The system was then preincubated at 30°C for 10 mln and the reaction initiated in a cuvette by addition of 20 /A of 7 mM t-butylhydroperoxlde and 200 /zl of N A D P H in 10 m M T r l s - H C l buffer, pH 7 0. Decrease in absorbance at 340 nm was monitored at 30°C. Glutathione S-transferase (GST) activity was measured according to the method of Hablg et al. [17] using l-chloro-2,4-dinatrobenzene (CDNB)
44 as the substrate. All e n z y m a t i c assays were c o n d u c t e d with a H~tachl 557 d o u b l e b e a m s p e c t r o p h o t o m e t e r . G S H a n d G S S G c o n t e n t was m e a s u r e d following the m e t h o d o f H i s s m a n d H d f [ 1 8 ] . P r o t e i n c o n t e n t in the m l c r o s o m a l a n d cytosolic fractions was d e t e r m i n e d by the m e t h o d o f L o w r y et al. [19] with bovine serum a l b u m i n as the s t a n d a r d .
Statzsttcal analysts D a t a were a n a l y z e d for m e a n s a n d s t a n d a r d d e v i a t i o n (S.D.) a n d c o m p a r e d by the S t u d e n t ' s t-test. T h e level o f slgmficance for all e x p e r i m e n t s was set at P < 0.05. Results
B o d y a n d o r g a n weights following D M F a d m l m s t r a t l o n for 1 week are shown in T a b l e I. B o d y weight gain in the D M F - t r e a t e d g r o u p was the same as that o f the c o n t r o l group, but hver weights were significantly greater m the D M F g r o u p Spleen weights in the c o n t r o l a n d D M F - t r e a t e d g r o u p s were essentially the same T h e effects o f D M F a d m i n i s t r a t i o n for 1 week on h e m a t o l o g i c a l p a r a m e t e r s are examined. The two g r o u p s showed no differences ( d a t a n o t shown). S e r u m chemistry values for the two g r o u p s are shown In T a b l e I1. G O T and G P T increased in the D M F - t r e a t e d g r o u p , as did also c h o h n e s t e r a s e a n d total cholesterol T a b l e III shows the c o n t e n t o f hepatic m l c r o s o m a l protein, c y t o c h r o m e P-450, b5 a n d p r o t o h e m e , a l o n g with the activity o f N A D P H - c y t o c h r o m e c reductase and N A D H - f e r r l c y a m d e reductase after D M F t r e a t m e n t . H e p a t i c c y t o c h r o m e P-450, p r o t o h e m e a n d N A D H - f e r r l c y a n i d e reductase were slgmficantly decreased by 34"/,,, 24% a n d 24%, respectively, while there was no c h a n g e in hepatic m l c r o s o m a l protein, c y t o c h r o m e b5 a n d N A D P H - c y t o c h r o m e c reductase T a b l e IV indicates t h a t G S H a n d G S S G c o n t e n t a n d G P x activity were not affected by D M F a d m i n i s t r a t i o n , while G R activity was significantly decreased by 26% a n d G S T activity was slgmficantly increased by 66% at I week. Effects o f the d u r a t i o n o f D M F e x p o s u r e on G R , G P x a n d G S T in the hver are shown in F~g. 1. G R activity s h o w e d a significant decrease after the first injection TABLE I EFFECTS OF N,N-DIMETHYLFORMAMIDE ON BODY AND ORGAN WEIGHTS The results are expressed as mean 4- S D of 8 rats
Final bodywt (g) Lwer (g) (mg/g body wt) Spleen
Control
Exposed
2650
2540
",- 140
916 ± 109 34 48 + 2 85
+ 87
1032 4- 070" 40 62 4- 2 38**
(g)
0 6 8 4- 0 0 7
0 6 2 4- 0 0 7
(mg/g body wt)
2 55 -4- 0 17
2 45 4- 0 28
Slgmficantly &fferent from the control *P < 0 05, **P < 0 001
45
TABL E II E F F E C T S OF N , N - D I M E T H Y L F O R M A M I D E ON S ER U M C H E M I S T R Y The results are expressed as mean 4- S D of 8 samples Control Total protein (g/dl) Albumin (g/dl) Total bdlrubm (mg/dl) Glutamlc oxaloacetic transammase (Karmen umt) Glutamlc pyruv~c transammase (Karmen unit) Alkaline phosphatase (King-Armstrong Unit) Lactate dehydrogenase (Wrobleuskl Unit) Chollnesterase (IU) Total cholesterol (mg/dl) Trlglycerlde (mg/dl)
5 2 0 78
8 2 2 3
4444-
Exposed 0 1 02 00 70
5 8 4- 0 2 244-01 0 2 4- 0 0 216 5 4- 72 2**
23 6 4- 2 2
878 4- 33 7**
461 4 4- 82 0
397 9 4- 56 0
308 3 4- 133 0
5950 4- 3850
Significantly different from the control *P < 005
501 4- 15 4
723 4- 170"
72 5 4- 5 5 43 4 4- 10 0
1 1 8 4 4- 16 3**
415 4- 208
**P < 0 001
and remained depressed throughout the study, with no change in GPx activity GST activity was increased by 43"/,, at 3 days after treatment and thereafter timedependently. The time course of changes in GSH content were examined in the hvers after a single administration of a large dose of DMF. D M F caused an approximately 30% depletion of GSH at 8 h. GSH content recovered gradually with time, resuming the normal level at 24 h (Fig. 2). TABLE III EFFECTS OF N , N - D I M E T H Y L F O R M A M I D E O X Y G E N A S E SYSTEM
ON
HEPATIC
MICROSOMAL
The results are expressed as mean 4- S D of 8 samples Control Mlcrosomal protein (mg/g liver) Cytochrome P-450 (nmol/mg protein) Cytochromeb 5 (nmol/mg protein) Protoheme (nmol/mg protein) NADPH-cytochrome c reductase (nmol/mg protein per mln) NADH-ferrlcyamde reductase (u,mol/mg protein per mm)
26 1 4- 2 6 0 53 + 0 04
Exposed 23 8 4- 1 9 0 35 4- 0 04*
014 + 002
012 ± 001
0 90 4- 0 I 1 51 3 4- 9 7
0 68 • 0 06* 42 6 4- 8 0
Szgnlficantly different from the control *P < 0 001
I 96 4- 0 16
1 49 4- 0 21"
MONO-
46
TABLE IV EFFECTS OF N , N - D I M E T H Y L F O R M A M I D E
ON HEPATIC G L U T A T H I O N E METABOLISM
The results are expressed as mean :t: S D of 8 samples Control Reduced glutathlone
Exposed
8 6 5 6 -4- 6 0 7
7632 + 1103
260 2 -4- 69 8
273 4 -4- 68 4
720 + 66
5 3 0 -4- 6 3 *
367 9 :i: 59 2
374 8 :t: 34 5
999 4 :t: 53 6
1657 8 :i: 79 6*
(/~g/g hver)
Oxidized glutathlone (/~g/g hver)
Glutathlone reductase (nmol NADPH/mln per mg protein) Glutathlone perox]dase (nmol N A D P H / m m per mg protein) Glutathione-S-transferase (nmol GSH conjugated/mm per mg protein)
S]gmficantly different from the control *P < 0 001
GR achvdy
c_ o
[]
control
]
exposed
GPx activity 600
GST actlvrty
[]
control
[]
control
]
exposed
[]
exposed
Y~
E
~ 1500
'I- -J-
-F _T_.
c so
z
ill day
3 day
7
i
day
E E
200
c
0
day
3 day
7
day
I day
3 day
7 day
Fig 1 Effects of duration of dimethylformamlde (DMF) exposure on glutathlone reductase (GR), glutathlone peroxldase (GPx), glutathlone S-transferase (GST) an rat hver D M F was administered to the rats by subcutaneous mject~on at 0 5 ml/kg body wt dally for 1 week The control group was treated slmdarly with an equal volume of 0 9% NaCI Significantly different from control *P < 0 01, **P < 0 001
47
100
0
90 o o
~
80
(3)
=)
Z 0
7_ 7 o II: Z w 0
13)
6O
(7)
Z
0 0
50
0
2
4
6
TIME
8
12
AFTER
24
TREATMENT [hr]
Fig 2 Time course of changes m hepatxc GSH level in rats after dlmethylformamlde (DMF) exposure D M F was administered to the rats by subcutaneous reJect]on at 4 ml/kg body wt The control group was treated s]mtlarly w~th an equal volume of 0 9% NaCl Rats were k]lled at the mdmated times on the absossa All results are expressed as means of percentage decreases from controls The number of rats m each group ]s g]ven m parentheses Slgmficantly &fferent from control *P < 0 01, **P < 0 001
Discussion
Since the report by Smyth et al. in 1948 [20], consaderable research has been conducted on the toxicity of D M F in experimental animals The principal toxioty of D M F is hepatotoxlcity [21-25]. In humans, occupational exposure to D M F causes hepatic damage, intolerance to alcohol and abnormal gastrointestinal symptoms (e.g. nausea, vomiting, appetite loss, and abdominal pain) [4-7]. A previous report [26] also indicated depressed body weight gain after 2 weeks when rats were exposed to D M F by inhalation (1200 ppm). In the present study, control and exposed group rats were pmr-fed to minlm]ze differences due to &etary intake. Mild swelling was observed xn the liver and liver weight was increased in the DMF-treated group without depressed body weight gain. The increase in liver wexghts is considered to be a normal phenomenon (physiological adaptat]on) required for the biotransformation of DMF. Spinazzola et al. [27] noted the activity of aldolase, GOT, GPT, ALP and particularly sorbitol dehydrogenase (SDH) to increase following a single s c. administration of 2 ml/kg D M F to the rabbits. Scadteur et al. [28] found SDH not to increase after a single i.p. dose to the rats but clearly to be enhanced after four successive administrations of 0.5 ml/kg or 1.0 mi/kg. In the present study, serum GOT and GPT
48 increased after seven successive s c administrations o f 0 5 ml/kg Administration at the same dose for 3 days also caused the increase o f G O T and G P T (data not shown). Transammases, however, showed no increase by a single administration (0 5 ml/kg) (data not shown) As for hepatic mlcrosomal c y t o c h r o m e P-450, Scallteur et al [29] reported that in rats, a single or four times repeated 1.p. administrations o f 1 ml/kg D M F fa~led to have any effect on the concentration o f c y t o c h r o m e P-450 and h 5 In rats and humans, D M F is said to be metabolized by the hepatic mlcrosomal c y t o c h r o m e P-450. In the present study, seven repeated s c administrations o f 0.5 ml/kg D M F in rats caused a slgmficant decrease in c y t o c h r o m e P-450 content by 34% In our prehmlnary study, hepatic mlcrosomal c y t o c h r o m e P-450 did not decrease after a single injection at the same dose The cause o f c y t o c h r o m e P-450 depletion by D M F may possibly be due to (1) mlcrosomal P-450 destruction, (2) inhibition o f the synthesis of protoheme and apoproteln, or its promotion, (3) massive cellular damage, or damage to mlcrosomal membranes in the present study, cytochrome/)5, N A D P H cytochome c reductase were not affected So, we think that the cause of P-450 depletion may not be (3). C y t o c h r o m e P-450 cholesterol 7c~-hydroxylase (P-450 ch 7c0 is a biosynthetic P-450 enzyme that catalyzes the first and rate-limiting steps m the hepatic conversion o f cholesterol to bile acids [30]. In the present study, serum T-cho was observed to Increase significantly This finding suggests that P-450 ch 7c~ activity may be decreased by D M F . Recently, A M C C was confirmed to be present in the urine after exposure to D M F in humans and rodents [9] This Indicates that D M F metabohtes may possibly conjugate with G S H But, there have been few reports on the effect o f D M F on hepatic G S H Scallteur et al [31] could find no change in hepatic G S H concentration at 1 or 24 h after a single 1 p admmlstratlon o f D M F (1 0 ml/kg) G S H is present in high concentrations in most living cells [10] It is said that the depletion o f G S H may be of short duration, since G S H synthesis increases rapidly after acute depletion [11]. So, there IS the possibility that G S H content in the liver may resume the control level at 24 h after D M F injection Thus, the time-course of changes m hepatic G S H content and glutathlone metabohzlng enzymes was followed GST activity in the hver increased by 66% c o m p a r e d with that o f the control at 1 week after treatment (0 5 ml/kg) G S T may be an enzyme that detoxlfies D M F metabolltes conjugated with G S H in the liver Indeed, at 6 - 8 h after a single s.c administration of D M F (4 ml/kg), G S H in the liver was decreased by approximately 30% Transient decrease in G S H content in the liver Indicates that D M F metabohtes possibly undergo G S H conjugation The G S H redox cycle m which G R and GPx participate is considered to be Important in protecting tissues from oxidative stress [32]. In the present study, D M F inhibited G R activity even after a single lnjectmn of 0.5 ml/kg, while GPx actlwty was not affected t h r o u g h o u t the study. However, G S H and G S S G content in the liver did not change significantly at 1 week after D M F treatment. These results may imply a requirement for continuous regeneration of G S H not by G S H redox cycle, but by a de novo synthesis and/or other system [331. In conclusion, the repeated administration of D M F induced a decrease in hepatic cytochrome P-450 content and an increase in GST activity in the liver A smgle lnjec-
49
tton of D M F caused a transient decrease in hepatic G S H These results ln&cate that D M F alters the hepatic mlcrosomal monooxygenase system and glutathlone metabolism Further study should be conducted to clarify the mechantsm of hepatotoxlclty by DMF.
Acknowledgments We are grateful to Miss Norlko Yamada, Mtss Hlroko Ishtmoto and Mtss Yoshiko Yasumura for their skillful technical assistance
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30
31 32 33
O.H Lowry, N J Rosebrough, A L Farr and R J Randall, Protein measurement with the fohn phenol reagent J Blol C h e m , 193 (1951) 265 H F. Smyth and C P Carpenter, Further experience w,th the range finding test m the mdustr,al toxicology laboratory J lnd Hyg, 30 (1948) 63 H ltoh, T Uchlkoshl and K Olkawa, H~stopathologlcal mvesngatlon ofDMF-mduced hepatotox1city Acta Pathol. J p n , 37 (1987) 1879 K Tanaka, Toxicity ofdlmethyl-formam~de (DMF) to the young female rat lnt Arch. Arbeltsmed, 28 (1971) 95 V Scaflteur and R Lauwerys, Dlmethyl-formamlde (DMF) Hepatotoxlclty Toxicology, 43 (1987) 231 1 Lundberg, S Lundberg and T Kronevl, Some observations on dlmethylformamlde hepatotoxlclty Toxicology, 22 (1981) 1 G L Kennedy and H Sherman, Acute and subchromc toxicity of d,methylformamlde and dlmethylacetamlde followmg various routes of administration Drug Chem Toxtcol, 9 (1986) 147 D K Craig, R J We~r, W Wagner and D Groth, Subchromc inhalation toxicity of dlmethylformamlde m rats and mice Drug Chem Toxlcol, 7 (1984) 551 A Spmazzola, G Devoto, S Zedda, G Carta, G Satta, M T Slngu, S Cudom and S Zmnl, IntOSSlCazlone spenmentale da dlmetflformamlde Foha M e d , (1969) 739 V Scallteur, Contnbuuon a l'etude de la relation toxlclte -- metabohsme de la dlmethylformam~de chez le rat, Ph D Thesis, Umvers~te Cathohque de Louvam, Louvam, Belgmm, 1984 V Scaflteur, J P Buchet and R Lauwerys, The relationship between dlmethylformamlde metabohsm and toxicity, m S S Brown and D.S Dawes (Eds), Organ-Directed Toxicity, Chemical Indices and Mechamsms, Pergamon Press, Oxford 1981, p 169 D J Waxman, Rat hepatic cholesterol 7c~-hydroxylase Biochemical propemes and comparison to constltutwe and xenob~otlc-mduclble cytochrome /'-450 enzymes Arch Blochem Blophy, 247 (1986) 335 V Scaflteur and R Lauwerys, In wvo metabohsm of dlmethylformam~de and relationship to tox~c,ty m the male rat Arch Toxlcol, 56 (1984) 87 D J Reed, Regulation of reductlve processes by glutathlone Baochem Pharmacol, 35 (1986) 7 B H Lauterburg and J R Mitchell, Regulanon of hepanc glutathlone turnover m rats m wvo and ewdence for kinetic homogeneity of the hepatic glutathlone pool J Chn Invest, 67 (1981) 1415
41
Effects of dimethylformamide on hepatic microsomal monooxygenase system and glutathione metabolism in rats Kazuhiko Imazu a'b, Kazuya Fujishiro b and Naohide Inoue b aThtrd Department of Internal Medwme, School of Medtcme and bDepartment of Envtronmental Toxwology, lnstttute of lndustrtal Ecologwal Scwnce, Umverstty of Occupatzonal and Envtronmental Health 1-1 lsetgaoka Yahatantshtku, Kttalyushu 807 (Japan) (Recewed August 23rd, 1991, accepted December 8th, 1991)
Summary The effects of repeated exposure to N,N-&methylformamlde (DMF) on hepatic mlcrosomal monoox~genase system and glutathlone metabohsm were investigated D M F was administered to W~star male rats by subcutaneous (s c ) rejection at 0 5 ml/kg body weight dally for I week Macroscoplcally, mdd hver swelhng was observed and hver weights slgmficantly increased after 1 week of exposure to DMF Hematologxcal changes were not detected In exposed rats, glutamlc oxaloacetlc transammase, glutamlc pyruvlc transammase, chohnesterase and total cholesterol s~gmficantly increased Hepatic m~crosomal cytochrome P-450 and protoheme decreased by 34% and 24%, respectwely, while mlcrosomal protein and cytochrome b 5 were not affected NADH-ferrlcyamde reductase actwlty decreased by 24% whde NADPH-cytochrome c reductase actwlty showed no change Glutathlone reductase (GR) actwlty showed a slgmficant decrease after the first rejection and remamed depressed throughout the study, with no change m glutath~one peroxldase (GPx) actwlty Glutath~one S-transferase (GST) actwlty showed a slgmficant mcrease at 3 days after D M F treatment and gradually increased by 66% at 1 week In a subsequent experiment with a single administration of D M F (4 ml/kg), reduced glutathlone (GSH) m the hver was decreased by 28"/0 at 8 h, but recovered to control levels by 24 h These results indicate that DMF alters the hepatic mlcrosomal monooxygenase system and glutathlone metabohsm These findings may greatly contribute to the elucldaUon of the pathogenesls of DMF hepatotox~c~ty
Key words Dimethylformamlde, Liver, Cytochrome P-450, Glutathlone, Glutathlone S-transferase
Introduction N,N-dimethylformamide (DMF) is widely used as an organic solvent m laboratories and the chemical industry (e.g. in the manufacture of polymers such as polyvmylchlorlde, polyacrylonltrile and polyurethanes) [1]. It is absorbed through the lungs and very readily through the skin [2]. The primary target organ of D M F toxicity in experimental animals and humans is the liver [3,4]. Occupational exCorrespondence to Kazuhlko Imazu, Third Department of Internal Me&cme, School of Medicine. UOEH, 1-1 Iselgaoka Yahatamsh~ku, K~takyushu 807, Japan 0300-483X/92/$05.00 © 1992 Elsevier Scientific Pubhshers Ireland Ltd Printed and Pubhshed m Ireland
42 posure to D M F causes nausea, appetite loss, abdominal pare, alcohol intolerance and hepatic damage [4-7]. Very recently, Redlich et al [8] reported an outbreak of toxic liver disease among workers exposed to DMF. Little is known of its mechanism of hepatotoxicity. N-acetyl-S-(N-alkylcarbamoyl)cystelnes (AMCC) has been ldentiffed unequivocally in the urine after exposure to D M F in humans and rodents [9] This supports the possibility that conjugation with G S H is one metabolic pathway of D M F in addition to the classical metabolic pathway that D M F receives hydroxylation of one methyl group to yield N-hydroxymethyl-N-methylformamlde ( H M M F ) and N-demethylatlon to yield N-methylformamlde (NMF) and formamlde by cytochrome P-450. However, no detailed studies have been conducted on the effects of D M F on hepatic glutathlone metabolizing enzymes and mxcrosomal monooxygenase system. In this study, we investigated the effects of D M F on hepatic drug metabolizing enzymes, particularly the microsomal monooxygenase system, cytochrome P-450 and glutathione metabolism. Materials and methods
Ammals and chemicals Wistar male rats, approximately 7 weeks of age, were obtained from Selwa Experimental Animals Ltd. (Fukuoka, Japan) They were randomly divided into control and DMF-treated groups and were acclimated to the animal facility for 1 week prior to the experiment. The environmental conditions were a temperature of 24 ± I°C, relative humidity of 55 ± 5°C and a 12 h light-dark cycle (light from 07:00 to 19:00 h). The rats of the exposed group had free access to food (Japan CLEA, CE-2), while the control groups were given the same volume of food that the exposed groups took in the day before (pair-fed controls) Water was available ad hbitum. D M F (98% pure) was purchased from Wako Pure Chemical Industries L t d , Japan. All chemicals used were of reagent grade.
Expertmental destgn D M F was administered by subcutaneous (s.c.) xnlectlon at 0.5 ml/kg body wt (475 mg/kg) daily for 1 week. Control rats were treated with an equal volume of 0 9'/,, NaC1. Eight animals were used for each group Food was removed 24 h before killing. The animals were killed m pairs, one from each group, under deep ether anesthesia about 24 h after the last treatment. Blood was obtained from the subclavian artery with ethylenedlaminetetraacetlc acid, dipotasslum salt (EDTA-2K), as an anticoagulant for blood cell counts, or with heparin sodium salt for other assays. The livers were perfused in situ with cold 0.9% NaC1 and then liver and spleen were removed and weighed. A portion of the hver was homogenized with 3 vol 1.15% KC1 using a Potter-Elvejem homogenizer with a Teflon pestle. To an aliquot of the homogenate was added 1.0 ml metaphosphorlc acid, followed by centrlfugatlon at 10 0000 × g for 30 min in a Beckman L8-M ultracentrifuge The supernatant was used to determine reduced glutathaone (GSH) and oxidized glutathlone (GSSG) content. The rest of the homogenate was centrifuged at 10 000 × g for 20 min and the supernatant centrifuged at 105 000 × g for 60 man in a Beckman L 8 - 5 0 M / E ultracentrifuge. This supernatant, the cytosolic fraction, was used to determine
43 glutathione metabohzlng enzyme activities. The pellet containing the mlcrosomal fraction was used to determine cytochrome P-450, b5 and protoheme content. N A D P H - c y t o c h r o m e c reductase and NADH-ferrlcyanide reductase activity were also measured using the macrosomal fraction G S H is present an high concentrations in most living cells [10] It Is said that the depletion of G S H may be of short duration, since G S H synthesis increases rapidly after acute depletion [11]. So, there is the posslbihty that G S H content in the liver may resume the control level at 24 h after D M F injection. The second study was conducted to confirm the decrease of G S H in the liver. Exposed animals received 4 ml/kg (3800 mg/kg) D M F by s c route, and were killed at specified times (2, 4, 6, 8, 12 and 24 h) following treatment. The control group recexved 0 9°/,, NaCI, followlng the same dosing schedule. Food was removed after D M F injection. G S H content in each laver was determined as described above
Hematological and blochemtcal exammatton A Towa Counter Analyzer (Model E-3000) was used for white blood cell counts (WBC), red blood cell counts (RBC) and determlnatlon of hemoglobin concentration (Hb) and hematocrit (Ht) An Hitachi Counter Analyzer (Model H-710, 736) was used to determine total protein (TP), albumin (Alb), total blhrubin (T-Bll), glutamic oxaloacetic transamlnase (GOT), glutamlc pyruvic transammase (GPT), alkaline phosphatase (Alp), lactate dehydrogenase (LDH), chohnesterase (Ch-E), total cholesterol (T-cho) and triglycerlde (TG) Assay methods The concentration of cytochrome P-450 in laver mlcrosomes was determined from the reduced CO difference spectrum using an extinction coefficient of 91 m M -J cm -1 as reported by Omura and Sato [12]. Cytochrome b5 and protoheme content in the liver was estimated by the methods of Stnttmatter and Vehck [13] and Omura and Sato [12], respectively. N A D P H - c y t o c h r o m e c reductase and N A D H ferracyanide reductase activity were determined by the method of Philhps and Langdon [14]. Glutathione reductase (GR) activity was measured by the method of Carlberg and Mannervak [15] with mo&ficatlon. The reaction mixture contained 1.99 ml of 0.2 M potassium phosphate buffer, pH 7.0, with 2 mM E D T A and 10 ~1 of cytosol. A total of 200/A of GSSG was added to the mixture followed by incubation at 30°C for 10 min. The reaction was initiated an a cuvette by the addition of 200 tA of 2 m M reduced nlCotlnamlde adenine dinucleotlde phosphate ( N A D P H ) in 10 mM Tris-HC1 buffer, pH 7.0. Decrease In absorbance was momtored at 340 nm and 30°C Glutathione peroxidase (GPx) activity was measured by a slightly modified method of Beutler et al [16] The reaction mixture contained 200 tA of 1 M Tris-HC1 buffer, p H 8.0, 40/zl of 0.1 M GSH, 100/xl of 10 IU/ml GR, 100/zl of 1 M N a N 3, 5 #1 of cytosol and 1 vol. of water to bring the final volume to 1.78 ml. The system was then preincubated at 30°C for 10 mln and the reaction initiated in a cuvette by addition of 20 /A of 7 mM t-butylhydroperoxlde and 200 /zl of N A D P H in 10 m M T r l s - H C l buffer, pH 7 0. Decrease in absorbance at 340 nm was monitored at 30°C. Glutathione S-transferase (GST) activity was measured according to the method of Hablg et al. [17] using l-chloro-2,4-dinatrobenzene (CDNB)
44 as the substrate. All e n z y m a t i c assays were c o n d u c t e d with a H~tachl 557 d o u b l e b e a m s p e c t r o p h o t o m e t e r . G S H a n d G S S G c o n t e n t was m e a s u r e d following the m e t h o d o f H i s s m a n d H d f [ 1 8 ] . P r o t e i n c o n t e n t in the m l c r o s o m a l a n d cytosolic fractions was d e t e r m i n e d by the m e t h o d o f L o w r y et al. [19] with bovine serum a l b u m i n as the s t a n d a r d .
Statzsttcal analysts D a t a were a n a l y z e d for m e a n s a n d s t a n d a r d d e v i a t i o n (S.D.) a n d c o m p a r e d by the S t u d e n t ' s t-test. T h e level o f slgmficance for all e x p e r i m e n t s was set at P < 0.05. Results
B o d y a n d o r g a n weights following D M F a d m l m s t r a t l o n for 1 week are shown in T a b l e I. B o d y weight gain in the D M F - t r e a t e d g r o u p was the same as that o f the c o n t r o l group, but hver weights were significantly greater m the D M F g r o u p Spleen weights in the c o n t r o l a n d D M F - t r e a t e d g r o u p s were essentially the same T h e effects o f D M F a d m i n i s t r a t i o n for 1 week on h e m a t o l o g i c a l p a r a m e t e r s are examined. The two g r o u p s showed no differences ( d a t a n o t shown). S e r u m chemistry values for the two g r o u p s are shown In T a b l e I1. G O T and G P T increased in the D M F - t r e a t e d g r o u p , as did also c h o h n e s t e r a s e a n d total cholesterol T a b l e III shows the c o n t e n t o f hepatic m l c r o s o m a l protein, c y t o c h r o m e P-450, b5 a n d p r o t o h e m e , a l o n g with the activity o f N A D P H - c y t o c h r o m e c reductase and N A D H - f e r r l c y a m d e reductase after D M F t r e a t m e n t . H e p a t i c c y t o c h r o m e P-450, p r o t o h e m e a n d N A D H - f e r r l c y a n i d e reductase were slgmficantly decreased by 34"/,,, 24% a n d 24%, respectively, while there was no c h a n g e in hepatic m l c r o s o m a l protein, c y t o c h r o m e b5 a n d N A D P H - c y t o c h r o m e c reductase T a b l e IV indicates t h a t G S H a n d G S S G c o n t e n t a n d G P x activity were not affected by D M F a d m i n i s t r a t i o n , while G R activity was significantly decreased by 26% a n d G S T activity was slgmficantly increased by 66% at I week. Effects o f the d u r a t i o n o f D M F e x p o s u r e on G R , G P x a n d G S T in the hver are shown in F~g. 1. G R activity s h o w e d a significant decrease after the first injection TABLE I EFFECTS OF N,N-DIMETHYLFORMAMIDE ON BODY AND ORGAN WEIGHTS The results are expressed as mean 4- S D of 8 rats
Final bodywt (g) Lwer (g) (mg/g body wt) Spleen
Control
Exposed
2650
2540
",- 140
916 ± 109 34 48 + 2 85
+ 87
1032 4- 070" 40 62 4- 2 38**
(g)
0 6 8 4- 0 0 7
0 6 2 4- 0 0 7
(mg/g body wt)
2 55 -4- 0 17
2 45 4- 0 28
Slgmficantly &fferent from the control *P < 0 05, **P < 0 001
45
TABL E II E F F E C T S OF N , N - D I M E T H Y L F O R M A M I D E ON S ER U M C H E M I S T R Y The results are expressed as mean 4- S D of 8 samples Control Total protein (g/dl) Albumin (g/dl) Total bdlrubm (mg/dl) Glutamlc oxaloacetic transammase (Karmen umt) Glutamlc pyruv~c transammase (Karmen unit) Alkaline phosphatase (King-Armstrong Unit) Lactate dehydrogenase (Wrobleuskl Unit) Chollnesterase (IU) Total cholesterol (mg/dl) Trlglycerlde (mg/dl)
5 2 0 78
8 2 2 3
4444-
Exposed 0 1 02 00 70
5 8 4- 0 2 244-01 0 2 4- 0 0 216 5 4- 72 2**
23 6 4- 2 2
878 4- 33 7**
461 4 4- 82 0
397 9 4- 56 0
308 3 4- 133 0
5950 4- 3850
Significantly different from the control *P < 005
501 4- 15 4
723 4- 170"
72 5 4- 5 5 43 4 4- 10 0
1 1 8 4 4- 16 3**
415 4- 208
**P < 0 001
and remained depressed throughout the study, with no change in GPx activity GST activity was increased by 43"/,, at 3 days after treatment and thereafter timedependently. The time course of changes in GSH content were examined in the hvers after a single administration of a large dose of DMF. D M F caused an approximately 30% depletion of GSH at 8 h. GSH content recovered gradually with time, resuming the normal level at 24 h (Fig. 2). TABLE III EFFECTS OF N , N - D I M E T H Y L F O R M A M I D E O X Y G E N A S E SYSTEM
ON
HEPATIC
MICROSOMAL
The results are expressed as mean 4- S D of 8 samples Control Mlcrosomal protein (mg/g liver) Cytochrome P-450 (nmol/mg protein) Cytochromeb 5 (nmol/mg protein) Protoheme (nmol/mg protein) NADPH-cytochrome c reductase (nmol/mg protein per mln) NADH-ferrlcyamde reductase (u,mol/mg protein per mm)
26 1 4- 2 6 0 53 + 0 04
Exposed 23 8 4- 1 9 0 35 4- 0 04*
014 + 002
012 ± 001
0 90 4- 0 I 1 51 3 4- 9 7
0 68 • 0 06* 42 6 4- 8 0
Szgnlficantly different from the control *P < 0 001
I 96 4- 0 16
1 49 4- 0 21"
MONO-
46
TABLE IV EFFECTS OF N , N - D I M E T H Y L F O R M A M I D E
ON HEPATIC G L U T A T H I O N E METABOLISM
The results are expressed as mean :t: S D of 8 samples Control Reduced glutathlone
Exposed
8 6 5 6 -4- 6 0 7
7632 + 1103
260 2 -4- 69 8
273 4 -4- 68 4
720 + 66
5 3 0 -4- 6 3 *
367 9 :i: 59 2
374 8 :t: 34 5
999 4 :t: 53 6
1657 8 :i: 79 6*
(/~g/g hver)
Oxidized glutathlone (/~g/g hver)
Glutathlone reductase (nmol NADPH/mln per mg protein) Glutathlone perox]dase (nmol N A D P H / m m per mg protein) Glutathione-S-transferase (nmol GSH conjugated/mm per mg protein)
S]gmficantly different from the control *P < 0 001
GR achvdy
c_ o
[]
control
]
exposed
GPx activity 600
GST actlvrty
[]
control
[]
control
]
exposed
[]
exposed
Y~
E
~ 1500
'I- -J-
-F _T_.
c so
z
ill day
3 day
7
i
day
E E
200
c
0
day
3 day
7
day
I day
3 day
7 day
Fig 1 Effects of duration of dimethylformamlde (DMF) exposure on glutathlone reductase (GR), glutathlone peroxldase (GPx), glutathlone S-transferase (GST) an rat hver D M F was administered to the rats by subcutaneous mject~on at 0 5 ml/kg body wt dally for 1 week The control group was treated slmdarly with an equal volume of 0 9% NaCI Significantly different from control *P < 0 01, **P < 0 001
47
100
0
90 o o
~
80
(3)
=)
Z 0
7_ 7 o II: Z w 0
13)
6O
(7)
Z
0 0
50
0
2
4
6
TIME
8
12
AFTER
24
TREATMENT [hr]
Fig 2 Time course of changes m hepatxc GSH level in rats after dlmethylformamlde (DMF) exposure D M F was administered to the rats by subcutaneous reJect]on at 4 ml/kg body wt The control group was treated s]mtlarly w~th an equal volume of 0 9% NaCl Rats were k]lled at the mdmated times on the absossa All results are expressed as means of percentage decreases from controls The number of rats m each group ]s g]ven m parentheses Slgmficantly &fferent from control *P < 0 01, **P < 0 001
Discussion
Since the report by Smyth et al. in 1948 [20], consaderable research has been conducted on the toxicity of D M F in experimental animals The principal toxioty of D M F is hepatotoxlcity [21-25]. In humans, occupational exposure to D M F causes hepatic damage, intolerance to alcohol and abnormal gastrointestinal symptoms (e.g. nausea, vomiting, appetite loss, and abdominal pain) [4-7]. A previous report [26] also indicated depressed body weight gain after 2 weeks when rats were exposed to D M F by inhalation (1200 ppm). In the present study, control and exposed group rats were pmr-fed to minlm]ze differences due to &etary intake. Mild swelling was observed xn the liver and liver weight was increased in the DMF-treated group without depressed body weight gain. The increase in liver wexghts is considered to be a normal phenomenon (physiological adaptat]on) required for the biotransformation of DMF. Spinazzola et al. [27] noted the activity of aldolase, GOT, GPT, ALP and particularly sorbitol dehydrogenase (SDH) to increase following a single s c. administration of 2 ml/kg D M F to the rabbits. Scadteur et al. [28] found SDH not to increase after a single i.p. dose to the rats but clearly to be enhanced after four successive administrations of 0.5 ml/kg or 1.0 mi/kg. In the present study, serum GOT and GPT
48 increased after seven successive s c administrations o f 0 5 ml/kg Administration at the same dose for 3 days also caused the increase o f G O T and G P T (data not shown). Transammases, however, showed no increase by a single administration (0 5 ml/kg) (data not shown) As for hepatic mlcrosomal c y t o c h r o m e P-450, Scallteur et al [29] reported that in rats, a single or four times repeated 1.p. administrations o f 1 ml/kg D M F fa~led to have any effect on the concentration o f c y t o c h r o m e P-450 and h 5 In rats and humans, D M F is said to be metabolized by the hepatic mlcrosomal c y t o c h r o m e P-450. In the present study, seven repeated s c administrations o f 0.5 ml/kg D M F in rats caused a slgmficant decrease in c y t o c h r o m e P-450 content by 34% In our prehmlnary study, hepatic mlcrosomal c y t o c h r o m e P-450 did not decrease after a single injection at the same dose The cause o f c y t o c h r o m e P-450 depletion by D M F may possibly be due to (1) mlcrosomal P-450 destruction, (2) inhibition o f the synthesis of protoheme and apoproteln, or its promotion, (3) massive cellular damage, or damage to mlcrosomal membranes in the present study, cytochrome/)5, N A D P H cytochome c reductase were not affected So, we think that the cause of P-450 depletion may not be (3). C y t o c h r o m e P-450 cholesterol 7c~-hydroxylase (P-450 ch 7c0 is a biosynthetic P-450 enzyme that catalyzes the first and rate-limiting steps m the hepatic conversion o f cholesterol to bile acids [30]. In the present study, serum T-cho was observed to Increase significantly This finding suggests that P-450 ch 7c~ activity may be decreased by D M F . Recently, A M C C was confirmed to be present in the urine after exposure to D M F in humans and rodents [9] This Indicates that D M F metabohtes may possibly conjugate with G S H But, there have been few reports on the effect o f D M F on hepatic G S H Scallteur et al [31] could find no change in hepatic G S H concentration at 1 or 24 h after a single 1 p admmlstratlon o f D M F (1 0 ml/kg) G S H is present in high concentrations in most living cells [10] It is said that the depletion o f G S H may be of short duration, since G S H synthesis increases rapidly after acute depletion [11]. So, there IS the possibility that G S H content in the liver may resume the control level at 24 h after D M F injection Thus, the time-course of changes m hepatic G S H content and glutathlone metabohzlng enzymes was followed GST activity in the hver increased by 66% c o m p a r e d with that o f the control at 1 week after treatment (0 5 ml/kg) G S T may be an enzyme that detoxlfies D M F metabolltes conjugated with G S H in the liver Indeed, at 6 - 8 h after a single s.c administration of D M F (4 ml/kg), G S H in the liver was decreased by approximately 30% Transient decrease in G S H content in the liver Indicates that D M F metabohtes possibly undergo G S H conjugation The G S H redox cycle m which G R and GPx participate is considered to be Important in protecting tissues from oxidative stress [32]. In the present study, D M F inhibited G R activity even after a single lnjectmn of 0.5 ml/kg, while GPx actlwty was not affected t h r o u g h o u t the study. However, G S H and G S S G content in the liver did not change significantly at 1 week after D M F treatment. These results may imply a requirement for continuous regeneration of G S H not by G S H redox cycle, but by a de novo synthesis and/or other system [331. In conclusion, the repeated administration of D M F induced a decrease in hepatic cytochrome P-450 content and an increase in GST activity in the liver A smgle lnjec-
49
tton of D M F caused a transient decrease in hepatic G S H These results ln&cate that D M F alters the hepatic mlcrosomal monooxygenase system and glutathlone metabolism Further study should be conducted to clarify the mechantsm of hepatotoxlclty by DMF.
Acknowledgments We are grateful to Miss Norlko Yamada, Mtss Hlroko Ishtmoto and Mtss Yoshiko Yasumura for their skillful technical assistance
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9
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