LNVIRONIIIEN’IAL
RLSEtR(‘H
90-99
15,
Comparative
(1978)
Toxicity
of Trivalent Chromium
Alterations
S.K.
D.K.
TANDON,’
Irrdustritrl
To.~ico/ogy
in Blood
J.S.
SAXENA,
Resctrrch
C‘erltw.
Received
and Hexavalent
and Liver
GAUR.
AND
PO.\/ BO.Y No.
November
SATYA
V.
CHANDRA
80. L1r~.hrron~-226001.
Itltiitr
16. 1976
The effects of trivalent and hexavalent chromium compounds on rabbits were studied with a view toward investigating the toxic potentials of two different forms of chromium to which industrial workers or miners might be exposed. While both forms of chromium altered the levels of certain important chemical constituents of blood and serum and produced significant morphological changes in the liver, the hexavalent form induced a greater effect. The accumulation of the metal was also higher in animals exposed to chromium in oxidation state 6 than in those exposed to metal in state 3.
INTRODUCTION
Industrial applications of the compounds of chromium and the health hazards associated with them are already recognised (Walsh, 1953; Baetjer er al., 1974a,b: Royle, 1975). In these, chromium mainly occurs in two oxidation states, viz., trivalent and hexavalent, and both occur in environments of a number of occupations (Mancuso and Hueper, 1951). Although, trivalent chromium compounds have lesser applications, their toxic potential cannot be ignored especially in view of the possibility of the reduction of the hexavalent to the trivalent form in the biological system and the subsequent formation of stable complexes with the body proteins (Hueper and Payne, 1959; Barborik, 1970). Therefore, it was of interest to study the comparative toxicity of two forms of chromium to which chromite miners, chromate workers, or consumers of the products of this industry might be exposed. In order to have a preliminary assessment of this sort, the effects of chromium nitrate (trivalent) and potassium dichromate (hexavalent) on the morphology of liver and on the levels of certain chemical constituents of blood in relation to the concentration of chromium were investigated in experimental rabbits. MATERIALS
AND METHODS
Male albino rabbits from Industrial Toxicology Research Centre’s colony, weighing around 1.5 kg and maintained on cd libitlrm standard pellet diet, were used in the investigation. They were divided into three groups. two comprising ten animals each, and the third consisting of eight animals. The animals of groups I and II were injected ip daily for 6 weeks with 2 mg of Cr/kg, as chromium nitrate and as potassium dichromate (E. Merck, G.R.) dissolved in 1 ml of 0.9% sodium chloride, respectively. Group III received an equal Anin~uls
crtzd treatment.
’ To whom
requests
for reprints
should
be addressed. 90
0013.9351/78/0151LO090$0?.00/0 Copyright All
rights
cl
1978
of reproductmn
by
Academic in
Press. any
form
Inc. reserved.
CHROMIL’hl
TOXICITY
IS
RABBITS
91
volume of normal saline and served as control. The animals were weighed every week and the dose was adjusted accordingly. Five animals from each of groups I and II and four from group III were sacrificed at 3- and 6-week intervals, 72 hours after the last injection. The 72-hour period was allowed to enable excretion of most of the unbound chromium from the system. The liver was removed immediately and washed free of adherent material. The blood was collected from ear veins in fluoride tubes for blood sugar, in oxalated tubes for blood urea, and in centrifuge tubes for separating serum. Studies OH blood rrrld .serlllrz. Standard procedures were adopted for estimation of sugar (Folin and Wu. 1920) and urea (Levine et (I(., 196 I) in blood; total and free cholesterol (Wooton, 1964), total phospholipids (Folch et al., 1957), ascorbic acid (Schaffert and Kingsley, 1955), and total protein (Lowry et ul., 1951) were determined in serum. Hemlttohgy. The total and differential white blood cells were counted and the blood hemoglobin was estimated according to Sahli’s method (Dacie and Lewis, 1966). Estirnutiort oj‘chtmmitrm. The metal was estimated in liver, blood, and serum spectrophotometrically (Gooderson and Salt, 1968) following wet ashing of the samples. Histological techiylres. Liver from control and experimental rabbits was fixed in 10% neutral buffered formalin. After routine processing, the tissue was embedded in paraffin. and sections were cut at 5 pm and stained with hematoxylin-eosin. RESULTS
There was no mortality or morbidity among animals of all the three groups, which apparently remained healthy throughout the course of the experiment. On gross examination, the liver was found to be markedly congested in experimental animals. The levels of some important chemical constituents of blood and serum and the morphological gradings in liver together with the concentration of chromium in control and experimental rabbits are recorded in Tables 1 and 2, respectively. Biochrmiccrl Studies The level of urea increased significantly and that of sugar remained practically within the normal range in blood of rabbits treated with either the trivalent or the hexavalent chromium. While the increase in the urea level was more marked at 6 weeks than at 3 weeks of treatment. there was only a slight increase in the content of chromium from 3 to 6 weeks in both experimental groups. It is noteworthy that the concentration of chromium was far higher in blood of animals injected with hexavalent chromium than in those treated with the trivalent form. The total and free cholesterol levels in the serum remained almost within the normal range in all experimental rabbits. On the other hand. serum total phospholipids showed a sharp decrease at 3-weeks and a marked increase at 6 weeks. the effects being comparatively more prominent in the case of the hexavalent form. The level of serum protein increased significantly and that of ascorbic acid remained unaltered
92
TANDON
TABLE
ET AL.
1
BLOOD
ASD SERUM CHESIISTRP AFTER DAILY ADIV~INISI.RATION OP CHROMII‘M NI I-RAT-E (TRIVAI.ENT CHROMIVX~) AND POTASSIUM DICHROMA.I.E (HEXAVALENT CHROMIURI) TO RABBITS FOK 3 .AND 6 WEEKS”
Trivalent
Blood Sugafl tmg/lOO ml) Urea (mgi100 ml) Chromium (FCLpiml) Serum Total cholesteroP (mgi100 ml) Free cholesterolb tmg/lOO ml) Total phospholipids tmg/lOO ml) Total protein (g/100 ml) Ascorbic acid @g/ml) Chromium t&ml) (I Each experimental * Within c Based * P < ** P
RLSEtR(‘H
90-99
15,
Comparative
(1978)
Toxicity
of Trivalent Chromium
Alterations
S.K.
D.K.
TANDON,’
Irrdustritrl
To.~ico/ogy
in Blood
J.S.
SAXENA,
Resctrrch
C‘erltw.
Received
and Hexavalent
and Liver
GAUR.
AND
PO.\/ BO.Y No.
November
SATYA
V.
CHANDRA
80. L1r~.hrron~-226001.
Itltiitr
16. 1976
The effects of trivalent and hexavalent chromium compounds on rabbits were studied with a view toward investigating the toxic potentials of two different forms of chromium to which industrial workers or miners might be exposed. While both forms of chromium altered the levels of certain important chemical constituents of blood and serum and produced significant morphological changes in the liver, the hexavalent form induced a greater effect. The accumulation of the metal was also higher in animals exposed to chromium in oxidation state 6 than in those exposed to metal in state 3.
INTRODUCTION
Industrial applications of the compounds of chromium and the health hazards associated with them are already recognised (Walsh, 1953; Baetjer er al., 1974a,b: Royle, 1975). In these, chromium mainly occurs in two oxidation states, viz., trivalent and hexavalent, and both occur in environments of a number of occupations (Mancuso and Hueper, 1951). Although, trivalent chromium compounds have lesser applications, their toxic potential cannot be ignored especially in view of the possibility of the reduction of the hexavalent to the trivalent form in the biological system and the subsequent formation of stable complexes with the body proteins (Hueper and Payne, 1959; Barborik, 1970). Therefore, it was of interest to study the comparative toxicity of two forms of chromium to which chromite miners, chromate workers, or consumers of the products of this industry might be exposed. In order to have a preliminary assessment of this sort, the effects of chromium nitrate (trivalent) and potassium dichromate (hexavalent) on the morphology of liver and on the levels of certain chemical constituents of blood in relation to the concentration of chromium were investigated in experimental rabbits. MATERIALS
AND METHODS
Male albino rabbits from Industrial Toxicology Research Centre’s colony, weighing around 1.5 kg and maintained on cd libitlrm standard pellet diet, were used in the investigation. They were divided into three groups. two comprising ten animals each, and the third consisting of eight animals. The animals of groups I and II were injected ip daily for 6 weeks with 2 mg of Cr/kg, as chromium nitrate and as potassium dichromate (E. Merck, G.R.) dissolved in 1 ml of 0.9% sodium chloride, respectively. Group III received an equal Anin~uls
crtzd treatment.
’ To whom
requests
for reprints
should
be addressed. 90
0013.9351/78/0151LO090$0?.00/0 Copyright All
rights
cl
1978
of reproductmn
by
Academic in
Press. any
form
Inc. reserved.
CHROMIL’hl
TOXICITY
IS
RABBITS
91
volume of normal saline and served as control. The animals were weighed every week and the dose was adjusted accordingly. Five animals from each of groups I and II and four from group III were sacrificed at 3- and 6-week intervals, 72 hours after the last injection. The 72-hour period was allowed to enable excretion of most of the unbound chromium from the system. The liver was removed immediately and washed free of adherent material. The blood was collected from ear veins in fluoride tubes for blood sugar, in oxalated tubes for blood urea, and in centrifuge tubes for separating serum. Studies OH blood rrrld .serlllrz. Standard procedures were adopted for estimation of sugar (Folin and Wu. 1920) and urea (Levine et (I(., 196 I) in blood; total and free cholesterol (Wooton, 1964), total phospholipids (Folch et al., 1957), ascorbic acid (Schaffert and Kingsley, 1955), and total protein (Lowry et ul., 1951) were determined in serum. Hemlttohgy. The total and differential white blood cells were counted and the blood hemoglobin was estimated according to Sahli’s method (Dacie and Lewis, 1966). Estirnutiort oj‘chtmmitrm. The metal was estimated in liver, blood, and serum spectrophotometrically (Gooderson and Salt, 1968) following wet ashing of the samples. Histological techiylres. Liver from control and experimental rabbits was fixed in 10% neutral buffered formalin. After routine processing, the tissue was embedded in paraffin. and sections were cut at 5 pm and stained with hematoxylin-eosin. RESULTS
There was no mortality or morbidity among animals of all the three groups, which apparently remained healthy throughout the course of the experiment. On gross examination, the liver was found to be markedly congested in experimental animals. The levels of some important chemical constituents of blood and serum and the morphological gradings in liver together with the concentration of chromium in control and experimental rabbits are recorded in Tables 1 and 2, respectively. Biochrmiccrl Studies The level of urea increased significantly and that of sugar remained practically within the normal range in blood of rabbits treated with either the trivalent or the hexavalent chromium. While the increase in the urea level was more marked at 6 weeks than at 3 weeks of treatment. there was only a slight increase in the content of chromium from 3 to 6 weeks in both experimental groups. It is noteworthy that the concentration of chromium was far higher in blood of animals injected with hexavalent chromium than in those treated with the trivalent form. The total and free cholesterol levels in the serum remained almost within the normal range in all experimental rabbits. On the other hand. serum total phospholipids showed a sharp decrease at 3-weeks and a marked increase at 6 weeks. the effects being comparatively more prominent in the case of the hexavalent form. The level of serum protein increased significantly and that of ascorbic acid remained unaltered
92
TANDON
TABLE
ET AL.
1
BLOOD
ASD SERUM CHESIISTRP AFTER DAILY ADIV~INISI.RATION OP CHROMII‘M NI I-RAT-E (TRIVAI.ENT CHROMIVX~) AND POTASSIUM DICHROMA.I.E (HEXAVALENT CHROMIURI) TO RABBITS FOK 3 .AND 6 WEEKS”
Trivalent
Blood Sugafl tmg/lOO ml) Urea (mgi100 ml) Chromium (FCLpiml) Serum Total cholesteroP (mgi100 ml) Free cholesterolb tmg/lOO ml) Total phospholipids tmg/lOO ml) Total protein (g/100 ml) Ascorbic acid @g/ml) Chromium t&ml) (I Each experimental * Within c Based * P < ** P
