Madhusudan G. Soni

Adenosine triphosphate protection of chlordecone-amplified CCl4 hepatotoxicity and lethality

Dietary exposure to a nontoxic level of chlordecone (10 ppm for 15 days) followed by a single exposure to a subtoxic dose of CCl4 (100 microliters/kg, ip) is known to result in a 67-fold amplification of CCl4 toxicity. The hypothesis that the underlying mechanism is due to incapacitation of hepatocytes leading to an ablation of the early-phase hormetic response of tissue repair as a consequence of precipitous decline in hepatic glycogen and ATP, received experimental support from Mehendale in 1990. The present study was designed to investigate if direct administration of ATP to rats maintained on the chlordecone diet would result in protection from the hepatotoxic and lethal effects of the chlordecone+CCl4 combination. Male Sprague-Dawley rats (125-150 g) were maintained either on a diet containing no added contaminants (control) or on a diet containing 10 ppm chlordecone for 15 days, and were challenged with CCl4 (100 microliters/kg, ip) on day 16. Without ATP administration all rats died within 72 h, while administration of ATP (100 mg/rat, sc) to chlordecone-pretreated rats at -1, +1, 3, 5, 12, 24 and 36 h of CCl4 injection resulted in 100% survival. Injection of ATP, at -1, +1, 3 and 5 h of CCl4 administration to chlordecone pretreated rats decreased plasma enzyme elevations (alanine and aspartate aminotransferase, sorbitol dehydrogenase) as well as substantially preventing elevation of plasma bilirubin levels at 6, 12 and 24 h. Hepatic ATP levels were also elevated at 6 and 12 h, but not at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue Repair Response as a Function of Dose during Trichloroethylene Hepatotoxicity

Trichloroethylene (TCE), a widely used organic solvent and degreasing agent, is regarded as a hepatotoxicant. The objective of the present studies was to investigate whether the extent and timeliness of tissue repair has a determining influence on the ultimate outcome of hepatotoxicity. Male Sprague-Dawley rats (200-250 g) were injected with a 10-fold dose range of TCE and hepatotoxicity and tissue repair were studied during a time course of 0 to 96 h. Light microscopic changes as evaluated by H&E-stained liver sections revealed a dose-dependent necrosis of hepatic cells. Maximum liver cell necrosis was observed at 48 h after the TCE administration. However, liver injury as assessed by plasma sorbitol dehydrogenase (SDH) showed a dose response over a 10-fold dose range only at 6 h, whereas alanine aminotransferase (ALT) did not show a dose response at any of the time points studied. A low dose of TCE (250 mg/kg) showed an increase in SDH at all time points up to 96 h without peak levels, whereas higher doses showed peak only at 6 h. At later time points SDH declined but remained above normal. In vitro addition of trichloroacetic acid, a metabolite of TCE to plasma, decreased the activities of SDH and ALT indicating that metabolites formed during TCE toxicity may interfere with plasma enzyme activities in vivo. This indicates that the lack of dose-related increase in SDH and ALT activities may be because of interference by the TCE metabolite. Tissue regeneration response as measured by [3H]thymidine incorporation into hepatocellular nuclear DNA was stimulated maximally at 24 h after 500 mg/kg TCE administration. A higher dose of TCE led to a delay and diminishment in [3H]thymidine incorporation. At a low dose of TCE (250 mg/kg) [3H]thymidine incorporation peaked at 48 h and this could be attributed to very low or minimal injury caused by this dose. With higher doses tissue repair was delayed and attenuated allowing for unrestrained progression of liver injury. These results support the concept that the toxicity and repair are opposing responses and that a dose-related increase in tissue repair represents a dynamic, quantifiable compensatory mechanism.

Extent and Timeliness of Tissue Repair Determines the Dose-Related Hepatotoxicity of Chloroform

As a part of mixture toxicity studies, the objective of the present investigation was to validate the hypothesis that the rate and extent of liver tissue repair response to a given dose determines the end result of toxicity (death or recovery), regardless of the mechanisms by which injury is inflicted, using a well-known environmental pollutant, chloroform (CHCl3). In future, the data will be used to compare with the results of mixtures containing CHCl3 to aid in characterizing the safety of chemical mixtures and to construct a physiologically based pharmacokinetic (PBPK) model for dose, route, and species extrapolation. Hepatotoxicity and tissue repair were measured in male Sprague-Dawley rats (S–D) receiving a 10-fold dose range of CHCl3 (74, 185, 370, and 740 mg/kg, IP) during a time course of 0 to 96 hours. Liver injury, as assessed by plasma alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) elevation, increased with dose over the 10-fold dose range. Because CHCl3 is also known to cause kidney damage, blood urea nitrogen (BUN) and creatinine were measured to evaluate the kidney injury. With doses up to 370 mg/kg, liver injury increased in a dose-related fashion, which peaked at 24 hours and returned to normal after 48 hours, whereas at highest dose (740 mg/kg), the injury was progressive resulting in 90% mortality. Blood and liver CHCl3 levels were quantified using gas chromatography (GC) over a time course of 30 to 360 minutes. The dose-related increase in the blood and liver CHCl3 levels were consistent with dose-dependent liver injury. Tissue regeneration response, as measured by [3H]-thymidine incorporation into hepatocellular nuclear DNA peaked at 36 hours in rats treated with the lower two doses of CHCl3 (74 and 185 mg/kg). Further increase in CHCl3 dose to 370 mg/kg resulted in an earlier increase in [3H]-thymidine incorporation at 24 hours, which peaked at 36 hours. However, at the highest dose of CHCl3 (740 mg/kg), tissue repair was delayed and attenuated, allowing for unrestrained progression of liver injury. The kidney injury markers after CHCl3 administration were not different from controls. These results support the concept that in addition to the magnitude of tissue repair response, the time at which this response occurs is critical in restraining the progression of injury. Measuring tissue repair and injury as simultaneous biological responses to toxic agents might increase the usefulness of dose-response paradigms in predictive toxicology and risk assessment. Although the dosimetry of the present study was well beyond the environmental exposure levels of CHCl3, a PBPK model will be developed in future based upon these data to evaluate the effects at environmental levels.

Hepatic failure leads to lethality of chlordecone-amplified hepatotoxicity of carbon tetrachloride

Chlordecone (Kepone) amplification of CCl4 toxicity occurs at small, nontoxic levels of chlordecone and CCl4 and results in highly increased irreversible hepatotoxicity culminating in lethality. Although it is generally assumed that CCl4 lethality is due to hepatic failure, no definitive studies are available in the literature bridging massive liver failure and death. The present studies were designed to evaluate whether hepatic failure is the cause of the lethality during chlordecone-amplified CCl4 toxicity. Male Sprague-Dawley rats were maintained on control or a chlordecone (10 ppm) diet for 15 days and injected with CCl4 (100 microliters/kg, ip) on Day 16. Rats were killed at 0, 6, 12, 24, 36, and 48 hr after CCl4 challenge. Hepatic failure was evaluated by measuring plasma glucose, ammonia, bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), sorbitol dehydrogenase (SDH), hepatic ATP, glycogen, and by histological and histomorphometric analyses. Plasma creatinine, urea, and kidney histopathology were also assessed for possible renal injury. As expected CCl4 administration to chlordecone-pretreated rats resulted in 20% lethality by 36 hr, which progressed with time, and all rats died within 72 hr. A significant and progressive hypoglycemia was observed with a 60% reduction in plasma glucose at 48 hr. Hepatic glycogen content dropped precipitously. Similarly, hepatic ATP levels remained suppressed (80% of control) at all the time points studied. Plasma ammonia levels were significantly elevated, and by 48 hr, a threefold increase was observed. Plasma ALT, AST, SDH, and bilirubin increased progressively until the death of rats receiving the chlordecone + CCl4 combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatoprotective agent (+)-cyanidanol increases the synthetic phase of hepatocellular regeneration

1. (+)-Cyanidanol (250 mg/kg) administration to male rats resulted in a concentration-dependent increase in [3H]-thymidine incorporation into hepatic nuclear DNA as well as a corresponding increase in the per cent of labelled cells. 2. The increase in [3H]-thymidine incorporation and per cent labelled cells was significant by 24 hr, maximal between 48 and 96 hr, and declined very slowly to normal by 15 days (360 hr). 3. Administration of (+)-cyanidanol resulted in an increase in heptic putrescine levels and ornithine decarboxylase activity at 6 hr but not at 24 hr. However, S-adenosylmethionine decarboxylase and spermidine acetyltransferase activities were unaltered. 4. Inspite of these favorable conditions, for cell division, mitotic index (per cent cells in metaphase) was not increased by (+)-cyanidanol. 5. These results along with previous findings indicate that (+)-cyanidanol stimulates the S-phase activity of hepatocellular regeneration, but the commitment to M-phase depends on the occurrence of liver injury.