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Monitoring chromium nephrotoxicity
102
Occupational health
Fd ('osmet. Toxicol. Vol. lS, No. 1
Monitoring chromium nephrotoxicity Franchini, 1.. Mutti, A., Cavatorta, A., Corradi. A., Cosi, A., Olivetti, G. & Borghetti, A, 11978). Nephrotoxicity of chromium. Remarks on an experimental and epidemiological investigation. Contr. Nephrol. IlL 98. Previous experimental studies with rats have shown thai in chromate nephrotoxicity the amount of urinary lysozyme increases as damage to the proximal tubule becomes more marked (Cited in F.C.T. 1976. 14, 71t. Using this and other urinary indicators of renal-tubule damage, an attempt was made in the above-cited study' to define more closely, the mechanisms responsible for the toxic effects of chromium in rats and to relate them to obserwltions made on a group of chromium-exposed workers. hi an acute study of chromium toxicity,, female Wistar rats received potassium dichromate in a single sc rejection {15 mg,kg body weight) and the effects were determined during three periods after dosing 11 S, 8 24 arrd 24 48 hr/. Tile glomerular filtration rate remained constant and within normal limits throughout the observation period. The chromium concentrations m the serum ullrafihrate and urine were considerabl~ higher than those found in control subjects and were progressively reduced, Chromium clearance and the excretion fraction were signiticantly higher than m the controls and remained constant throughout the 48 hr. In addition, tile median values for urinary indicators of cellular lesions (fl-glucuronidase) and of altered tubular metabolism {proteinuria, lysozymuria and glucosuria) were signilicantl? higher than m tile controls and increased progressively. The functional alterations observed corresponded to various renal morphological changes: there was a gradual appearance of cytoplasmic vacuolization, and " complete cellular necrosis was observed 24 hr after dosing. In a more prolonged study. 24 female Wistar rats were given 3rag potassium dichromate,kg body, weight everx other day for 8 wk, and the renal-function indices were determined weekly. There was a progressive increase in the amount of chromium in the serum ultrafiltrate and an increase m the quantit,¢ of chromium excreted in the urine over the treatment period. This was accompanied by' a significant in-' crease in chromium clearance and in the fraction of the chromium filtrate excreted. There was also a close correlation between the clearance of chromium and tile concentration of the metal in the cortical region of the kidney'. No clear relationship was established between increasing degrees of intoxication and the severity of tubular damage, as indicated by' proteinuria, urinar~ l~-glucuronidase and lysozymuria, although these were higher on average than in the control animals and the fl-glucuronidase levels were intermittently higher even in the early stages of the study. The levels of fl-glucuronidase activit2, proteinuria and lysozymuria determined in three groups of chromium-exposed workers were to some extent comparable with the experimental findings. The first group had had only limited exposure {while welding on stainless steel) and showed no evidence of any change in renal function. In the other two groups, who had
had greater chromium exposure {in electrode welding on armoured steel and chromium plating), there were strong indications of tubular damage and, in particular, the fi-glucnronidase evidence was positive. The electrophoretic pattern of the urinary proteins was also indicativc of tubular damage. These effects seem likely to reflect a direct action on the epithelium of the proximal tubule and to be reversible following cessation of exposure and repair of the epithelial lesion.
Microsomal chromate metabolism Gruber, J. E. & Jennette, K. W. 119781. Metabolism of the carcinogen chromate by rat liver microsomes. Biochem. hiophy~. Res. Commun. 82, 700. Epidemiological studies have shown an association between exposure to chromium compounds and lung cancer (Cited i~1 F.C.T. 1979, 17, 97). Although most of these studies have implicated hexavalent chromium t(Trv') compounds there is no conclusive evidence for a particular carcinogenic chromium compound (Sunderman, Prevent. Med. 1976. 5, 279j. The metabolism m citro of ('r ~ b~ rat-liver microsomes has now been studied and it is proposed lhat the ultimate carcinogen of chromiuna may be tile trivalent (Or m) form. Microsomal fractions were prepared from tile livers of male Sprague Dawley rats that had been given an ip injection of 2(1 mg sodium dichromate:kg 16 19 hr before they were killed, The microsomes were incubated with 4 x I(} 4M-chromate (from potassium dichromate) in (I.t)5 M-TRIS. HCI buffer at pH 74 and diP,i:rent concentrations of N A D P H were added. Samples were taken a! various times, tile reaction was stopped by' protein extraction and the amounts of Cr vI. Cr m and NA D PH were measured spectrophotometrically, The incubation of chromate with rat-liver microseines resulted in the reduction of Crv' to Cr m. The amount of chromate reduced depended on the concentration of the microsomal preparation and the concentration of N A D P H . The maximum conversion occurred when 1.44 × I0 3M-NADPH was incubated with 15 mg microsomes nd for 6 hr. No reduction occurred in the absence of NADPH, and only a ver\ slow' rate of reduction occurred in the absence of microsomes. The results indicate, therefore, that an NADPH-requirmg enzyme or enzyme system is revolved in the metabolism of chromate m citr,. The authors propose that Cr"' is ultimately responsible for the carcinogenic effects of chromium. They base this theory on earlier epidemiological and animal studies and on mutagcnicity assays, h~ vitro studies have demonstrated thai Cr xt call easily cross cell membranes but Cr m cannot. In cellular assay systems. ('r'" has been shown to be mutagenic only at concentrations ver\ much [about 10 a times) greater than CrY( However in a sub-cellular system, Cr "~ proved to be mutagenic at concentrations much lower than Crv'. The hypothesis put forward is that Cr w crosses the cell membrane and may then be reduced to Cr m by a microsomal-enzyme system The Cr ul is capable of binding to proteins and nucleic acids and so may have mutagenic and carcinogenic effects. [-There has been another report of the metabolism
Occupational health
Fd ('osmet. Toxicol. Vol. lS, No. 1
Monitoring chromium nephrotoxicity Franchini, 1.. Mutti, A., Cavatorta, A., Corradi. A., Cosi, A., Olivetti, G. & Borghetti, A, 11978). Nephrotoxicity of chromium. Remarks on an experimental and epidemiological investigation. Contr. Nephrol. IlL 98. Previous experimental studies with rats have shown thai in chromate nephrotoxicity the amount of urinary lysozyme increases as damage to the proximal tubule becomes more marked (Cited in F.C.T. 1976. 14, 71t. Using this and other urinary indicators of renal-tubule damage, an attempt was made in the above-cited study' to define more closely, the mechanisms responsible for the toxic effects of chromium in rats and to relate them to obserwltions made on a group of chromium-exposed workers. hi an acute study of chromium toxicity,, female Wistar rats received potassium dichromate in a single sc rejection {15 mg,kg body weight) and the effects were determined during three periods after dosing 11 S, 8 24 arrd 24 48 hr/. Tile glomerular filtration rate remained constant and within normal limits throughout the observation period. The chromium concentrations m the serum ullrafihrate and urine were considerabl~ higher than those found in control subjects and were progressively reduced, Chromium clearance and the excretion fraction were signiticantly higher than m the controls and remained constant throughout the 48 hr. In addition, tile median values for urinary indicators of cellular lesions (fl-glucuronidase) and of altered tubular metabolism {proteinuria, lysozymuria and glucosuria) were signilicantl? higher than m tile controls and increased progressively. The functional alterations observed corresponded to various renal morphological changes: there was a gradual appearance of cytoplasmic vacuolization, and " complete cellular necrosis was observed 24 hr after dosing. In a more prolonged study. 24 female Wistar rats were given 3rag potassium dichromate,kg body, weight everx other day for 8 wk, and the renal-function indices were determined weekly. There was a progressive increase in the amount of chromium in the serum ultrafiltrate and an increase m the quantit,¢ of chromium excreted in the urine over the treatment period. This was accompanied by' a significant in-' crease in chromium clearance and in the fraction of the chromium filtrate excreted. There was also a close correlation between the clearance of chromium and tile concentration of the metal in the cortical region of the kidney'. No clear relationship was established between increasing degrees of intoxication and the severity of tubular damage, as indicated by' proteinuria, urinar~ l~-glucuronidase and lysozymuria, although these were higher on average than in the control animals and the fl-glucuronidase levels were intermittently higher even in the early stages of the study. The levels of fl-glucuronidase activit2, proteinuria and lysozymuria determined in three groups of chromium-exposed workers were to some extent comparable with the experimental findings. The first group had had only limited exposure {while welding on stainless steel) and showed no evidence of any change in renal function. In the other two groups, who had
had greater chromium exposure {in electrode welding on armoured steel and chromium plating), there were strong indications of tubular damage and, in particular, the fi-glucnronidase evidence was positive. The electrophoretic pattern of the urinary proteins was also indicativc of tubular damage. These effects seem likely to reflect a direct action on the epithelium of the proximal tubule and to be reversible following cessation of exposure and repair of the epithelial lesion.
Microsomal chromate metabolism Gruber, J. E. & Jennette, K. W. 119781. Metabolism of the carcinogen chromate by rat liver microsomes. Biochem. hiophy~. Res. Commun. 82, 700. Epidemiological studies have shown an association between exposure to chromium compounds and lung cancer (Cited i~1 F.C.T. 1979, 17, 97). Although most of these studies have implicated hexavalent chromium t(Trv') compounds there is no conclusive evidence for a particular carcinogenic chromium compound (Sunderman, Prevent. Med. 1976. 5, 279j. The metabolism m citro of ('r ~ b~ rat-liver microsomes has now been studied and it is proposed lhat the ultimate carcinogen of chromiuna may be tile trivalent (Or m) form. Microsomal fractions were prepared from tile livers of male Sprague Dawley rats that had been given an ip injection of 2(1 mg sodium dichromate:kg 16 19 hr before they were killed, The microsomes were incubated with 4 x I(} 4M-chromate (from potassium dichromate) in (I.t)5 M-TRIS. HCI buffer at pH 74 and diP,i:rent concentrations of N A D P H were added. Samples were taken a! various times, tile reaction was stopped by' protein extraction and the amounts of Cr vI. Cr m and NA D PH were measured spectrophotometrically, The incubation of chromate with rat-liver microseines resulted in the reduction of Crv' to Cr m. The amount of chromate reduced depended on the concentration of the microsomal preparation and the concentration of N A D P H . The maximum conversion occurred when 1.44 × I0 3M-NADPH was incubated with 15 mg microsomes nd for 6 hr. No reduction occurred in the absence of NADPH, and only a ver\ slow' rate of reduction occurred in the absence of microsomes. The results indicate, therefore, that an NADPH-requirmg enzyme or enzyme system is revolved in the metabolism of chromate m citr,. The authors propose that Cr"' is ultimately responsible for the carcinogenic effects of chromium. They base this theory on earlier epidemiological and animal studies and on mutagcnicity assays, h~ vitro studies have demonstrated thai Cr xt call easily cross cell membranes but Cr m cannot. In cellular assay systems. ('r'" has been shown to be mutagenic only at concentrations ver\ much [about 10 a times) greater than CrY( However in a sub-cellular system, Cr "~ proved to be mutagenic at concentrations much lower than Crv'. The hypothesis put forward is that Cr w crosses the cell membrane and may then be reduced to Cr m by a microsomal-enzyme system The Cr ul is capable of binding to proteins and nucleic acids and so may have mutagenic and carcinogenic effects. [-There has been another report of the metabolism