Trichloroethylene risk assessment: a review and commentary.

Trichloroethylene (TCE) is a widespread environmental contaminant that is carcinogenic when given in high, chronic doses to certain strains of mice and rats. The capacity of TCE to cause cancer in humans is less clear. The current maximum contaminant level (MCL) of 5 ppb (microg/L) is based on an US Environment Protection Agency (USEPA) policy decision rather than the underlying science. In view of major advances in understanding the etiology and mechanisms of chemically induced cancer, USEPA began in the late 1990s to revise its guidelines for cancer risk assessment. TCE was chosen as the pilot chemical. The USEPA (2005) final guidelines emphasized a "weight-of-evidence" approach with consideration of dose-response relationships, modes of action, and metabolic/toxicokinetic processes. Where adequate data are available to support reversible binding of the carcinogenic moiety to biological receptors as the initiating event (i.e., a threshold exists), a nonlinear approach is to be used. Otherwise, the default assumption of a linear (i.e., nonthreshold) dose-response is utilized. When validated physiologically based pharmacokinetic (PBPK) models are available, they are to be used to predict internal dosimetry as the basis for species and dose extrapolations. The present article reviews pertinent literature and discusses areas where research may resolve some outstanding issues and facilitate the reassessment process. Key research needs are proposed, including role of dichloroacetic acid (DCA) in TCE-induced liver tumorigenesis in humans; extension of current PBPK models to predict target organ deposition of trichloroacetic acid (TCA) and DCA in humans ingesting TCE in drinking water; use of human hepatocytes to ascertain metabolic rate constants for use in PBPK models that incorporate variability in metabolism of TCE by potentially sensitive subpopulations; measurement of the efficiency of first-pass elimination of trace levels of TCE in drinking water; and assessment of exogenous factors' (e.g., alcohol, drugs) ability to alter metabolic activation and risks at such low-level exposure.

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 groundwater 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.

Primaquine-induced hemolytic anemia: role of splenic macrophages in the fate of 5-hydroxyprimaquine-treated rat erythrocytes.

Primaquine-induced hemolytic anemia is known to result from premature sequestration of damaged (but intact) erythrocytes by the spleen. We have shown previously that a phenolic metabolite, 5-hydroxyprimaquine (5-HPQ), is a direct-acting hemolytic agent in rats, suggesting that 5-HPQ is a mediator of the hemolytic response to primaquine. To investigate the fate of erythrocytes in vivo after in vitro exposure to 5-HPQ, rat (51)Cr-labeled erythrocytes were incubated with hemolytic concentrations of 5-HPQ and then readministered intravenously to rats. The time course of loss of radioactivity from blood and uptake into the spleen and liver was measured. In rats given 5-HPQ-treated erythrocytes, an increased rate of removal of radioactivity from the circulation was observed as compared with the vehicle control. The loss of blood radioactivity was accompanied by a corresponding increase in radioactivity appearing in the spleen but not in the liver. When rats were pretreated with clodronate-loaded liposomes to deplete splenic macrophages, there was a decreased rate of removal of radioactivity from the circulation and a markedly diminished uptake into the spleen. A role for phagocytic removal of 5-HPQ-treated red cells was confirmed in vitro using the J774A.1 macrophage cell line. Furthermore, depletion of red cell GSH with diethyl maleate significantly enhanced in vitro phagocytosis of 5-HPQ-treated red cells. The data indicate that splenic macrophages are responsible for removing 5-HPQ-treated red cells and support the postulate that this metabolite is a contributor to the hemolytic anemia induced after administration of the parent compound.

Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents.

Lipid peroxidation and the accompanying translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer have recently been identified as key components of a signaling pathway for phagocytosis of apoptotic cells by macrophages. Drug-induced hemolytic anemia has long been known to be caused by an accelerated uptake of damaged (but intact) erythrocytes by macrophages in the spleen, and this process has been associated with enhanced formation of reactive oxygen species (ROS). However, the role of lipid peroxidation in hemolytic injury has remained unclear, and the effect of hemolytic agents on the distribution of PS in the erythrocyte membrane is unknown. The present studies were undertaken to determine whether lipid peroxidation and PS translocation could be detected in rat and human erythrocytes by three types of direct-acting hemolytic agents--dapsone hydroxylamine, divicine hydroquinone, and phenylhydrazine. 2',7'-Dichlorodihydrofluorescein diacetate was employed as a probe for intracellular ROS formation; lipid peroxidation was assessed by GC/MS analysis of F2-isoprostanes; and PS externalization was measured by annexin V labeling and the prothrombinase assay. The data confirmed that all three hemolytic agents generate ROS within erythrocytes under hemolytic conditions; however, no evidence for lipid peroxidation or PS translocation was detected. Instead, ROS production by these hemolytic agents was associated with extensive binding of oxidized and denatured hemoglobin to the membrane cytoskeleton. The data suggest that the transmembrane signal for macrophage recognition of hemolytic injury may be derived from oxidative alterations to erythrocyte proteins rather than to membrane lipids.

Primaquine-induced hemolytic anemia: role of membrane lipid peroxidation and cytoskeletal protein alterations in the hemotoxicity of 5-hydroxyprimaquine.

Primaquine-induced hemolytic anemia is a toxic side effect that is due to premature splenic sequestration of intact erythrocytes. Previous studies have suggested that a phenolic metabolite, 5-hydroxyprimaquine (5-HPQ), mediates primaquine hemotoxicity by generating reactive oxygen species (ROS) within erythrocytes that overwhelm antioxidant defenses. However, the nature of the oxidative stress is not understood, and the molecular targets, whether protein and/or lipid, are unknown. To investigate the mechanism underlying the hemolytic activity of 5-HPQ, we have examined the effect of hemolytic concentrations of 5-HPQ on ROS formation within rat erythrocytes using the cellular ROS probe, 2',7'-dichlorodihydrofluoresein diacetate. In addition, we examined the effect of 5-HPQ on membrane lipids and cytoskeletal proteins. The data indicate that 5-HPQ causes a prolonged, concentration-dependent generation of ROS within erythrocytes. Interestingly, 5-HPQ-generated ROS was not associated with the onset of lipid peroxidation or an alteration in phosphatidylserine asymmetry. Instead, 5-HPQ induced oxidative injury to the erythrocyte cytoskeleton, as evidenced by changes in the normal electrophoretic pattern of membrane ghost proteins. Immunoblotting with an anti-hemoglobin antibody revealed that these changes were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that cytoskeletal protein damage, rather than membrane lipid peroxidation or loss of phosphatidylserine asymmetry, underlies the process of removal of erythrocytes exposed to 5-HPQ.

Role of oxidant stress in lawsone-induced hemolytic anemia.

Lawsone (2-hydroxy-1,4-naphthoquinone) is the active ingredient of henna (Lawsonia alba), the crushed leaves of which are used as a cosmetic dye. Application of henna can induce a severe hemolytic anemia, and lawsone is thought to be the causative agent. Administration of lawsone to rats has been shown to induce a hemolytic response that is associated with oxidative damage to erythrocytes. However, direct exposure of isolated erythrocytes to lawsone did not provoke oxidative damage, suggesting that lawsone must undergo extra-erythrocytic bioactivation in vivo. In the present study, the survival of rat 51Cr-labeled erythrocytes in vivo after in vitro exposure to lawsone and its hydroquinone form, 1,2,4-trihydroxynaphthalene (THN) has been examined. Neither lawsone nor THN were directly hemolytic or methemoglobinemic, even at high concentrations (>3 mM). Lawsone had no effect on erythrocytic GSH levels, whereas THN (3 mM) induced a modest depletion (approximately 30%). Cyclic voltammetry revealed that the lack of hemotoxicity of lawsone was associated with a poor capacity to undergo redox cycling. In contrast, ortho-substituted 1,4-naphthoquinones without a 2-hydroxy group, such as 2-methyl- and 2-methoxy-1,4-naphthoquinone, were redox active, were able to deplete GSH, and were direct-acting hemolytic agents. An oxidant stress-associated hemolytic response to lawsone could be provoked, however, if it was incubated with GSH-depleted erythrocytes. The data suggest that lawsone is a weak direct-acting hemolytic agent that does not require extra-erythrocytic metabolism to cause hemotoxicity. Thus, the hemolytic response to henna may be restricted to individuals with compromised antioxidant defenses.

Induction of peroxisome proliferation in cultured hepatocytes by a series of halogenated acetates.

Trichloroacetate (TCA) and dichloroacetate (DCA) are hepatocarcinogenic metabolites of the environmental pollutant trichloroethylene (TCE) and are common water contaminants. Induction of peroxisome proliferation via activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) has been proposed as a mechanism for their hepatocarcinogenic action. However, it is unclear whether these compounds are direct ligands of PPARalpha or whether activation occurs by a ligand-independent process. The present studies were undertaken to determine whether a primary rat hepatocyte model system could be used to examine structure-activity relationships of haloacetates for the induction of peroxisomal palmitoyl-CoA oxidation. The haloacetates tested differed in both type (iodo, bromo, chloro and fluoro) and extent (mono, di and tri) substitution. Significant differences were observed in both potency and efficacy. Potency varied over about two orders of magnitude, in the order of mono > di = tri. Within the monohalo-substituted series, the order of potency was iodo > bromo > chloro, with the fluoro analog being essentially inactive. The monoiodo- and monobromo-derivatives showed significant induction at 50 and 100 microM, respectively, but cytotoxicity precluded obtaining full concentration-response curves. The dihalo- and trihalo-acetates had generally similar potency, and, with the exception of the diflouro- and dibromoacetates, showed a maximal induction of two- to three-fold. Difluoroacetate and dibromoacetate induced palmitoyl-CoA oxidation by nine- and six-fold, respectively, approaching the effectiveness of Wy-14,643 (50 microM) in this system. Of interest, the slopes of the concentration-dependence lines of the difluoro- and dibromo-acetates were markedly dissimilar from the other di- and tri-haloacetates, suggesting either a marked difference in the way they activate the PPARalpha receptor or a substantial difference in the way they are metabolized or transported by the hepatocytes.

Primaquine-induced hemolytic anemia: susceptibility of normal versus glutathione-depleted rat erythrocytes to 5-hydroxyprimaquine.

Primaquine is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. Methemoglobinemia and hemolytic anemia, however, are dose-limiting side effects of primaquine therapy. These hemotoxic effects are believed to be mediated by metabolites, although the identity of the toxic specie(s) and the mechanism underlying hemotoxicity have remained unclear. Previous studies showed that an N-hydroxylated metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline, was capable of mediating primaquine-induced hemotoxicity. The present studies were undertaken to investigate the hemolytic potential of 5-hydroxyprimaquine (5-HPQ), a phenolic metabolite that has been detected in experimental animals. 5-HPQ was synthesized, isolated by flash chromatography, and characterized by NMR spectroscopy and mass spectrometry. In vitro exposure of (51)Cr-labeled erythrocytes to 5-HPQ induced a concentration-dependent decrease in erythrocyte survival (TC(50) of ca. 40 microM) when the exposed cells were returned to the circulation of isologous rats. 5-HPQ also induced methemoglobin formation and depletion of glutathione (GSH) when incubated with suspensions of rat erythrocytes. Furthermore, when red cell GSH was depleted (>95%) by titration with diethyl maleate to mimic GSH instability in human glucose-6-phosphate dehydrogenase deficiency, a 5-fold enhancement of hemolytic activity was observed. These data indicate that 5-HPQ also has the requisite properties to contribute to the hemotoxicity of primaquine. The relative contribution of N-hydroxy versus phenolic metabolites to the overall hemotoxicity of primaquine remains to be assessed.

Primaquine-induced hemolytic anemia: formation of free radicals in rat erythrocytes exposed to 6-methoxy-8-hydroxylaminoquinoline.

Primaquine is an important antimalarial drug that is often dose-limited in therapy by the onset of hemolytic anemia. We have shown recently that an N-hydroxy metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), is a direct-acting hemolytic agent in rat red cells and that the hemolytic activity of this metabolite is associated with GSH oxidation and oxidative damage to both membrane lipids and skeletal proteins. To determine whether the formation of free radicals may be involved in this process, rat red cells (40% suspensions) were incubated with hemolytic concentrations of MAQ-NOH (150-750 microM) and examined by EPR spectroscopy using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) as a spin trap. Addition of MAQ-NOH to red cell suspensions containing 10 mM EMPO gave rise to an EPR spectrum with hyperfine constants consistent with those of an EMPO-hydroxyl radical adduct standard. Of interest, formation of EMPO-OH was constant for up to 20 min and dependent on the presence of erythrocytic GSH. Although no other radical adduct signals were detected in the cells by EPR, spectrophotometric analysis revealed the presence of ferrylhemoglobin, which indicates that hydrogen peroxide is generated under these experimental conditions. The data support the hypothesis that oxygen-derived and possibly other free radicals are involved in the mechanism underlying MAQ-NOH-induced hemolytic anemia.

Primaquine-induced hemolytic anemia: effect of 6-methoxy-8-hydroxylaminoquinoline on rat erythrocyte sulfhydryl status, membrane lipids, cytoskeletal proteins, and morphology.

Previous studies have shown that 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), an N-hydroxy metabolite of the antimalarial drug, primaquine, is a direct-acting hemolytic agent in rats. To investigate the mechanism underlying this hemolytic activity, the effects of hemotoxic concentrations of MAQ-NOH on rat erythrocyte sulfhydryl status, membrane lipids, skeletal proteins, and morphology have been examined. Treatment of rat erythrocytes with a TC(50) concentration of MAQ-NOH (350 microM) caused only a modest and transient depletion of reduced glutathione (GSH) (~30%), which was matched by modest increases in the levels of glutathione disulfide and glutathione-protein mixed disulfides. Lipid peroxidation, as measured by thiobarbituric acid-reactive substances and F(2)-isoprostane formation, was induced in a concentration-dependent manner by MAQ-NOH. However, the formation of disulfide-linked hemoglobin adducts on membrane skeletal proteins and changes in erythrocyte morphology were not observed. These data suggest that hemolytic activity results from peroxidative damage to the lipid of the red cell membrane and is not dependent on skeletal protein thiol oxidation. However, when red cell GSH was depleted (>90%) by titration with diethyl maleate, hemolytic activity of MAQ-NOH was markedly enhanced. Of interest, exacerbation of hemotoxicity was not matched by increases in lipid peroxidation, but by the appearance of hemoglobin-skeletal protein adducts. Collectively, the data are consistent with the concept that MAQ-NOH may operate by more than one mechanism; one that involves lipid peroxidation in the presence of normal amounts of erythrocytic GSH, and one that involves protein oxidation in red cells with low levels of GSH, such as are seen in individuals with glucose-6-phosphate dehydrogenase deficiency.