JoEllyn M. McMillan

Galactosamine hepatotoxicity: Effect of galactosamine on glutathione resynthesis in rat primary hepatocyte cultures

The effect of galactosamine on the resynthesis of glutathione in rat primary hepatocyte cultures was investigated. Cultured rat hepatocytes were treated with galactosamine (4 mM) 1.5 hr prior to concurrent with, or 1.5 hr after cell attachment; total cellular glutathione was then measured over time. Addition of galactosamine at any of these times suppressed methionine-enhanced glutathione resynthesis in the cultures after a lag period of about 120 min. The lag period was not due to slow uptake of galactosamine by the cultured cells, since cellular UTP levels fell to less than 10% of controls within 60 min, a time frame comparable to that observed in vivo. Neither was the lag period a result of interference with cellular uptake of methionine or with conversion of methionine to cysteine, since the phenomenon was observed regardless of whether methionine or cysteine was used to promote glutathione resynthesis. Addition of uridine, which protects against galactosamine hepatotoxicity in vivo by replenishing hepatic UTP levels, did not prevent the suppression of glutathione resynthesis. The data indicate that (a) galactosamine inhibits the time-dependent resynthesis of glutathione in primary hepatocyte cultures, (b) a lag period exists for this response, and (c) this effect is not directly related to depletion of cellular UTP stores.

Application of cryopreserved human hepatocytes in trichloroethylene risk assessment : Relative disposition of chloral hydrate to trichloroacetate and trichloroethanol

Background Trichloroethylene (TCE) is a suspected human carcinogen and a common ground-water contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. Methods The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. Results There was large interindividual variation in the Vmax values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in Vmax values among individuals, disposition of CH into the two competing pathways was relatively constant. Conclusions These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver.

The effect of the trichloroethylene metabolites trichloroacetate and dichloroacetate on peroxisome proliferation and DNA synthesis in cultured human hepatocytes.

Dichloroacetate (DCA) and trichloroacetate (TCA) are metabolites of the environmental contaminant trichloroethylene (TCE) that are thought to be responsible for its hepatocarcinogenicity in B6C3F1 mice. TCA and DCA induce peroxisomal proliferation and are mitogenic in rodent liver. The susceptibility of humans to TCA- and DCA-induced hepatocarcinogenesis is unknown. The current studies were aimed at using both primary and long-term human hepatocyte cultures to study the effects of TCA, DCA, and a potent peroxisome proliferator, WY-14,643, on peroxisomal activity and DNA synthesis in human hepatocytes. Peroxisome proliferation, as assessed by palmitoyl-CoA oxidation activity, was below the limit of detection in all human cell lines tested. However, the human cell lines did display small but significant increases in CYP450 4A11 levels following treatment with WY-14,643 (0.1 mmol/L), indicting that the CYP 4A11 gene may be regulated by peroxisome proliferator-activated receptor α in humans. Similarly to their effect in rodent hepatocyte cultures, TCA and DCA were not complete mitogens in human hepatocyte cultures. In fact, DNA synthesis tended to be significantly decreased following treatment of the cells with WY-14,643, TCA, or DCA. In contrast to rodent hepatocyte responses, TCA and DCA did not increase palmitoyl-CoA oxidation and caused a decrease in DNA synthesis in human hepatocyte cultures, suggesting that humans may not be susceptible to TCA- and DCA-induced hepatocarcinogenesis.

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression.

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C‐arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine‐induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine‐treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: lack of involvement in cytotoxicity.

Galactosamine hepatotoxicity in vivo has long been associated with rapid and extensive depletion of hepatic uridine nucleotides. Depletion of uridine nucleotides is considered to be causal in the toxicity, as evidenced by the protective effect of uridine administration. However, the exact mechanism of galactosamine‐induced hepatic necrosis is still unclear. We have previously shown that the addition of galactosamine to rat primary hepatocyte cultures dramatically decreases production of nitric oxide, as measured in the 24 hour culture medium. The present study investigates whether decreased nitric oxide production contributes to the toxicity of galactosamine in primary hepatocyte cultures. Similar concentration‐response curves were observed for the decrease in nitric oxide production and galactosamine cytotoxicity, raising the possibility that there is a similar mechanism for these effects. Suppression of NO synthesis was a direct effect of galactosamine, rather than an indirect effect due to loss of cells from the cultures. Both cytotoxicity and the decrease in nitric oxide production were attenuated by coaddition of 3 mM uridine. However, galactosamine cytotoxicity was not enhanced by prior inhibition of hepatocellular NO synthesis nor was it attenuated by maintenance of culture NO levels with molsidomine or diethylamine NONOate. These data do not support a role for decreased hepatocyte nitric oxide production in galactosamine hepatocyte toxicity.

Dichloroacetic acid induction of peroxisome proliferation in cultured hepatocytes

Trichloroethylene is a widespread industrial solvent and one of the most common environmental contaminants. Trichloroethylene causes hepatocarcinoma in the B6C3F1 mouse in a dose‐dependent manner. Trichloroethylenes hepatocarcinogenicity is thought to be mediated through its metabolites trichloroacetate and dichloroacetate. Although the mechanism of action is not well understood, hepatic tumors are thought to arise as a result of excessive peroxisome‐dependent active oxygen production or secondary to enhanced cell replication. The peroxisome proliferative activity of trichloroacetate has been replicated in cultured rodent hepatocytes, while that of dichloroacetate has not been demonstrated. The present experiments were designed to characterize the peroxisome proliferative response to dichloroacetate in hepatocyte cultures from male B6C3F1 mice and male Long Evans rats. The cultured hepatocytes were treated after attachment with 0.1, 0.5, 1.0, 2.0, or 4.0 mM dichloroacetate for 72 hours. Peroxisome proliferation was assessed by measuring palmitoyl‐CoA oxidation and by immunoquantitation of peroxisomal bifunctional enzyme. Palmitoyl CoA oxidation increased in a concentration‐dependent manner, with maximal induction of 5.5‐ and 5‐fold in mouse and rat hepatocytes, respectively, after treatment with 2.0 mM dichloroacetate. Peroxisomal bifunctional enzyme protein levels also increased in a concentration‐dependent manner in both rat and mouse hepatocytes in response to dichloroacetate exposure. These results indicate that the peroxisomal response observed in vivo in response to dichloroacetate administration can be reproduced in primary cultures of rat and mouse hepatocytes treated with dichloroacetate. Further studies using this model system will help elucidate mechanisms of dichloroacetate‐induced hepatocarcinogenesis.

Contribution of 3,4-Dichlorophenylhydroxylamine in Propanil-Induced Hemolytic Anemia

The methemoglobinemia and hemolytic anemia observed in experimental animals given aniline has been shown to be mediated by its N-oxidation metabolite, phenylhydroxylamine (Harrison, J.H. et al., 1987; Harrison, J.H. et al., 1986). The aniline derivative, propanil (3,4-dichloropropionanilide), is a widely used arylamide herbicide that has been shown to induce methemoglobinemia in experimental animals following conversion of the parent amide to one or more oxidized metabolites (Singleton, S > D. et al., 1973; Chow, A.Y.K. et al., 1975). Two methemoglobinemic metabolites of propanil have been identified, 3,4-dichlorophenylhydroxylamine (N-hydroxy-3,4-dichloroaniline) and 6-hydroxy-3,4-dichloroaniline (McMillan, D.C. et al., 1990). In view of the hemolytic activity of phenylhydroxylamine, we have examined the hemolytic potential of propanil and its metabolites in rats.