Mechanistic analysis of S-(1,2-dichlorovinyl)-L-cysteine-induced cataractogenesis in vitro.
Chronic exposure to low concentrations of the nephrotoxic cysteine conjugate S-(1,2-dichlorovinyl)-l-cysteine (DCVC) causes cataracts in mice. This study explored mechanisms of DCVC-induced cataractogenesis using explanted lenses from male Sprague-Dawley rats. Lenses placed in organ culture were exposed to 2.5 microM-1 mM DCVC for 24 hr. DCVC caused concentration and time-dependent changes in biochemical markers of toxicity (lenticular adenosine 5'-triphosphate (ATP) content, mitochondrial reduction of the tetrazolium dye MTT, and glutathione (GSH) content) at concentrations >/=25 microM. Lens clarity was adversely affected at concentrations >/=50 microM. Within 24 hr, 1 mM DCVC altered lens ATP content (-77 +/- 2%), mitochondrial MTT reduction (-40 +/- 3%), and GSH content (-19 +/- 4%) (percent difference from controls, p < 0.05). ATP was the most sensitive index of DCVC exposure in this model, while lens weight was not altered. The role of lenticular DCVC metabolism was investigated using the beta-lyase inhibitor aminooxyacetic acid (AOA) and the flavin monooxygenase (FMO) inhibitor methimazole (MAZ). AOA (1 mM) provided nearly complete protection from changes in biochemical parameters and lens transparency caused by DCVC, while MAZ (1 mM) provided only partial protection. The mitochondrial Ca2+ uniport inhibitor ruthenium red (30 microM) and the poly(ADP ribosyl)transferase inhibitor 3-aminobenzamide (3 mM) were only partially protective, whereas adverse changes in lens transparency and biochemical markers were not prevented by an antioxidant (2 mM dithiothreitol) or nontoxic transport substrates (200 microM probenecid or 10 mm phenylalanine, S-benzyl-L-cysteine or para-aminohippuric acid). Calpain inhibitors E64d (100 microM) and calpain inhibitor II (1 mM) were ineffective in preventing opacity formation caused by DCVC. In a small separate study, DCVC toxicity to explanted lenses from cynomologus monkeys was also ameliorated by coincubation with AOA. These results indicate that opacity formation by DCVC in rodent and primate lenses in vitro is primarily mediated via lenticular beta-lyase metabolism of DCVC to a reactive metabolite. Metabolism of DCVC by FMO and perturbations in mitochondrial calcium (Ca2+) homeostasis and increased poly(ADP-ribosylation) of nuclear proteins may play a limited role in opacity formation in vitro. However, opacity formation does not appear to be the result of oxidative stress or calpain activation. DCVC toxicity to the lens was not blocked with competitive inhibitors of the amino acid and organic anion transporters of DCVC as is found in the kidney.
Walsh Clang
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Pfizer Central Research, Drug Safety Evaluation, Groton, Connecticut 06340, USA.
Aleo
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D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D002386:Cataract / Q000139:chemically induced*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D005978:Glutathione / Q000032:analysis; D008297:Male; D008713:Methimazole / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D008713:Methimazole; C039961:S-(1,2-dichlorovinyl)cysteine; D000255:Adenosine Triphosphate; D005978:Glutathione; D003545:Cysteine