3
Kidney Toxicity and Cancer

This chapter reviews information on the effects of trichloroethylene on the kidney, with emphasis on information generated since the U.S. Environmental Protection Agency released its draft health risk assessment on this chemical (EPA 2001b). The review focuses on scientific issues raised during the review process that have relevance in carrying out a human health risk assessment. Studies published before the draft risk assessment are sometimes discussed to provide the context of current knowledge. Noncancer and cancer toxicity are addressed separately; toxic effects of trichloroethylene on the nephron tubule have been proposed to have a role in cancer development, functioning as a promoter. That role is considered later in this chapter.

ROLE OF METABOLISM IN RENAL EFFECTS

Trichloroethylene nephrotoxicity, like that of several haloalkenes, is associated with a multistep metabolic pathway that includes hepatic or renal glutathione S-conjugate formation, enzymatic hydrolysis of the glutathione S-conjugates to cysteine S-conjugates, and renal uptake of cysteine S-conjugates. It is generally accepted that the cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine is the penultimate nephrotoxicant. S-(1,2-Dichlorovinyl)-L-cysteine can undergo bioactivation by renal cysteine S-conjugate β-lyase to reactive species (Figure 3-1), whose reaction with cellular proteins is associated with cell damage and death (Dekant et al. 1987, 1991; Pähler et al. 1999). A second pathway of haloalkene S-conjugates’ bioactivation and toxification involving sulfoxidation of haloalkene cysteine and mercapturic acid conjugates has been identified (Sausen and



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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues 3 Kidney Toxicity and Cancer This chapter reviews information on the effects of trichloroethylene on the kidney, with emphasis on information generated since the U.S. Environmental Protection Agency released its draft health risk assessment on this chemical (EPA 2001b). The review focuses on scientific issues raised during the review process that have relevance in carrying out a human health risk assessment. Studies published before the draft risk assessment are sometimes discussed to provide the context of current knowledge. Noncancer and cancer toxicity are addressed separately; toxic effects of trichloroethylene on the nephron tubule have been proposed to have a role in cancer development, functioning as a promoter. That role is considered later in this chapter. ROLE OF METABOLISM IN RENAL EFFECTS Trichloroethylene nephrotoxicity, like that of several haloalkenes, is associated with a multistep metabolic pathway that includes hepatic or renal glutathione S-conjugate formation, enzymatic hydrolysis of the glutathione S-conjugates to cysteine S-conjugates, and renal uptake of cysteine S-conjugates. It is generally accepted that the cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine is the penultimate nephrotoxicant. S-(1,2-Dichlorovinyl)-L-cysteine can undergo bioactivation by renal cysteine S-conjugate β-lyase to reactive species (Figure 3-1), whose reaction with cellular proteins is associated with cell damage and death (Dekant et al. 1987, 1991; Pähler et al. 1999). A second pathway of haloalkene S-conjugates’ bioactivation and toxification involving sulfoxidation of haloalkene cysteine and mercapturic acid conjugates has been identified (Sausen and

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues FIGURE 3-1 Composite figure of metabolic pathways relevant to renal toxicity demonstrated in mammalian tissue (see text for references). Abbreviations: DCVC, S-(1,2-dichlorovinyl)-L-cysteine; DCVG, S-(1,2-dichlorovinyl)glutathione; DCVT, 1,2-dichlorovinylthiol; GST, glutathions S-transferase; NAcDCVC, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine; NAT, N-acetyl transferase; TCA, trichloroacetic acid; TCOH, trichloroethanol; THF, tetrahydrofolate. Elfarra 1991; Park et al. 1992; Lash et al. 1994; Werner et al. 1995a,b, 1996; Birner et al. 1998). Sulfoxidation of haloalkyl cysteine S-conjugates can constitute a toxification independent of β-lyase-mediated bioactivation (Lash et al. 1994; Werner et al. 1995a,b, 1996; Birner et al. 1998). Lash et al. (2000a,b) extensively reviewed biotransformation and bioactivation of trichloroethylene. Since then, there have been additional investigations of the renal metabolism and effects of trichloroethylene, some with a focus on sulfoxidation, as well as the sulfoxidation and toxicity of other haloalkyl nephrotoxicants (see below). The sulfoxidation and toxicity of trichloroethylene S-conjugates (involving hepatic or kidney microsomal sulfoxidation of cysteine and mercapturic acid conjugates) have been clearly established (Sausen and Elfarra 1991; Lash et al. 1994; Werner et al. 1996; Krause et al. 2003; Lash et al. 2003). The first report of enzymatic trichloroethylene S-conjugate sulfoxidation was by Ripp et al. (1997), who demonstrated rabbit liver microsomal sulfoxidation of S-(1,2-dichlorovinyl)-L-cysteine. Sulfoxidation was cata-

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues lyzed mainly by flavin monooxygenase, rather than by cytochrome P-450 (CYP450), and was specific for rabbit flavin monooxygenase-3 (Ripp et al. 1997). S-(1,2-Dichlorovinyl)-L-cysteine sulfoxidation was also catalyzed by human flavin monooxygenase-3 but not by other isoforms of flavin monooxygenase (Krause et al. 2003). Human liver microsomes also catalyzed S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation (Krause et al. 2003). Sulfoxidation was not detected with human kidney microsomes, although only one kidney sample was evaluated (Krause et al. 2003). The lack of metabolism was attributed to the low and variable concentrations of flavin monooxygenase-3 expression in kidney, which ranged from trace amounts to 1.3 pg/mg protein, compared with liver (Krause et al. 2003). S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide, whether formed in the liver and translocated to the kidney or potentially formed renally in situ, was considered to play a possible role in trichloroethylene nephrotoxicity (Krause et al. 2003). The mercapturic acid conjugates of dichlorovinyl cysteine, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine, also undergo sulfoxidation, as shown for rat liver microsomes (Werner et al. 1996). Unlike S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation, S-(1,2-dichlorovinyl)-L-cysteine mercapturate sulfoxidation was catalyzed mainly if not exclusively by CYP450, and a role for flavin monooxygenase was excluded. Specifically, rat liver microsomal N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation was catalyzed predominantly by CYP3A isoforms (Werner et al. 1996). Haloalkyl S-conjugates undergo sulfoxidation primarily in the liver. S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide was quantified after S-(1,2-dichlorovinyl)-L-cysteine incubation with microsomes from human liver but was not detected in microsomes from human kidney (Krause et al. 2003). Sulfoxidation of both S- and N-acetyl cysteine conjugates of cis- and trans-1,3-dichloropropene was detected in pig liver but not in rat kidney microsomes (Park et al. 1992). Nevertheless, mercapturate sulfoxidation by human kidney microsomes has been observed, albeit at rates much slower than for liver microsomes (Altuntas et al. 2004). Whether microsomes from human liver or kidney catalyze the sulfoxidation of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, and the relative activities, remains unknown. In general, S-conjugate sulfoxidation might be mediated by CYP or by flavin monooxygenase. For example, sulfoxidation of S-allyl-L-cysteine and S-benzyl-L-cysteine and, at a lower rate, S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2,2-trichlorovinyl)-L-cysteine, was catalyzed by flavin monooxygenases (Ripp et al. 1997; Krause et al. 2003). In contrast, sulfoxidation of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine, N-acetyl-S-(1,1-difluoro-2-fluoromethoxy-2-[trifluoromethyl]ethyl)-L-cysteine, and N-acetyl-S-(1-fluoro-

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues 2-fluoromethoxy-2-[trifluoromethyl]vinyl)-L-cysteine was catalyzed mainly by CYP450 (Werner et al. 1995a,b, 1996; Altuntas et al. 2004). The relative contribution of CYP450 and flavin monooxygenase toward cysteine S-conjugate S-oxidation depends on the conjugate structure. Generally, nucleophilic sulfur atoms are oxidized preferentially by flavin monooxygenase, whereas nonnucleophilic sulfur atoms are oxidized preferentially by CYP450 (Ripp et al. 1997; Damani and Houdi 1988). Thus, cysteine conjugates with more nucleophilic sulfur atoms (S-allyl-L-cysteine, S-benzyl-L-cysteine) were much better microsomal flavin monooxygenase substrates in human kidney and liver and in rabbit liver than were those with less nucleophilic sulfur atoms (the various haloalkyl cysteine and mercapturic acid conjugates) (Ripp et al. 1997; Krause et al. 2003). This is likely because the sulfur atoms of allyl and benzyl compounds are more nucleophilic than that of vinyl compounds and because flavin monooxygenases tend to oxidize strong nucleophiles (Damani and Houdi 1988). Lipophilicity might also affect haloalkene S-conjugate sulfoxidation by flavin monooxygenase. S-Benzyl-L-cysteine is relatively lipophilic, with a nucleophilic sulfur atom, and has been shown to be a selective substrate for flavin monooxygenase (Sausen et al. 1993). N-Acetyl-S-(1-fluoro-2-fluoromethoxy-2-[trifluoromethyl]vinyl)-L-cysteine has a vinylic sulfur atom as well as strong electron-withdrawing fluorine atoms, which make the sulfur atom much less nucleophilic than those of S-allyl-L-cysteine, S-benzyl-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(1,2,2-trichlorovinyl)-L-cysteine. N-Acetyl-S-(1,1-difluoro-2-fluoromethoxy-2-[trifluoromethyl]ethyl)-L-cysteine and N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-[trifluoromethyl]vinyl)-L-cysteine are less lipophilic than S-benzyl-L-cysteine, rendering them theoretically less susceptible to flavin monooxygenase sulfoxidation, potentially also explaining the lack of flavin monooxygenase activity toward their sulfoxidation. Rat liver microsomal N-acetyl-1,2-S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-2,2-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation was catalyzed predominantly, if not exclusively, by CYP3A isoforms (Werner et al. 1996). This conclusion was based on induction of sulfoxidation by phenobarbital and dexamethasone, inhibition by troleandomycin, and correlation with CYP3A activity. Indeed, CYP3A has been shown to be the predominant CYP isoform catalyzing the rat or human liver microsomal sulfoxidation of all haloalkyl mercapturic acid conjugates studied to date, including N-acetyl-1,2-S-(1,2-dichlorovinyl)-L-cysteine, N-acetyl-2,2-S-(1,2-dichlorovinyl)-L-cysteine, N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine, N-acetyl-S-(1,1-difluoro-2-fluoromethoxy-2-[trifluoromethyl]ethyl)-L-cysteine, and N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-[trifluoromethyl]vinyl)-L-cysteine (Werner et al. 1995a,b, 1996; Altuntas et al. 2004), which has been confirmed with cDNA-expressed CYP450s (Werner et al. 1995b; Altuntas et al. 2004). The role of CYP3A

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues in sulfoxidation, together with the polymorphic expression of CYP3A5 in humans, raises the possibility of pharmacogenetic differences in sulfoxidation and hence toxicity in persons exposed to trichloroethylene. Indeed, sulfoxidation of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(1,1-difluoro-2-fluoromethoxy-2-[trifluoromethyl]ethyl)-L-cysteine, and N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-[trifluoromethyl]vinyl)-L-cysteine was also catalyzed by expressed CYP3A5 as well as by CYP3A4 (Werner et al. 1995b; Altuntas et al. 2004). These studies showing CYP3A-catalyzed mercapturate sulfoxidation were performed in vitro. The first evidence for the role of CYP3A in any S-conjugate sulfoxidation in rats in vivo was recently obtained with a related haloalkene (Sheffels et al. 2004). Sulfoxidation of trichloroethylene S-conjugates can constitute a toxification pathway independent of β-lyase-mediated bioactivation (Sausen and Elfarra 1991; Lash et al. 1994; Werner et al. 1995a,b, 1996; Birner et al. 1998). Sulfoxides of trichloroethylene S-conjugates are stable but can react readily with nonprotein thiols. Thus, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-1,2-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide react spontaneously with glutathione as an electrophile and Michael acceptor (Sausen and Elfarra 1991; Ripp et al. 1997; Rosner and Dekant 1999). N-Acetyl-1,2-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide reactivity is greater than that of other mercapturate sulfoxides, including those of N-acetyl-2,2-S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide, and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, which react only slowly or require bioactivation by glutathione S-transferase for conjugate formation (Ripp et al. 1997; Rosner et al. 1998; Rosner and Dekant 1999). Toxicity of the S-conjugate sulfoxides of trichloroethylene, and other haloalkenes, has been evaluated in vitro and in vivo. Qualitatively, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide replicated the rat renal tubular cell injury also caused by S-(1,2-dichlorovinyl)-L-cysteine (Lash et al. 1994). Quantitatively, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide was significantly more nephrotoxic than S-(1,2-dichlorovinyl)-L-cysteine to isolated rat distal, but not proximal, tubular cells in vitro (Lash et al. 1994). Like S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, the sulfoxide of the mercapturate N-acetyl-1,2-S-(1,2-dichlorovinyl)-L-cysteine was significantly more cytotoxic than equivalent concentrations of 1,2-S-(1,2-dichlorovinyl)-L-cysteine in rat renal proximal tubular cells (Werner et al. 1996). Greater cytotoxicity in rat renal tubular cells of N-acetyl-2,2-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide, and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine compared with their corresponding mercapturic acids was also observed (Birner et al. 1995; Werner et al. 1996). In rats in vivo, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide caused the same type of renal proximal tubular cell histologic changes

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues as trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine (Lash et al. 1994). S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide, however, was significantly more nephrotoxic than S-(1,2-dichlorovinyl)-L-cysteine at equivalent doses (Lash et al. 1994). A recent investigation evaluated the effects of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide on human renal proximal tubular cells (Lash et al. 2003). S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide caused obvious morphologic abnormalities and cellular necrosis at concentrations as low as 10 µM. S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide also caused apoptosis. Apoptosis occurred rapidly and at low toxic concentrations, whereas necrosis occurred at later incubation times and at higher sulfoxide concentrations. Compared with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide caused greater and more rapid depletion of both ATP and cellular glutathione than S-(1,2-dichlorovinyl)-L-cysteine. Less apoptosis was observed with S-(1,2-dichlorovinyl)-L-cysteine sulfoxide than with S-(1,2-dichlorovinyl)-L-cysteine, which was attributed to the more rapid depletion of ATP. These results suggested a role for both S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine sulfoxide in human renal tubular cell toxicity. Other haloalkyl mercapturate sulfoxides demonstrate similar characteristics. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide was significantly nephrotoxic in rats in vivo (Birner et al. 1998). More recently, the effects of the cysteine-S-, mercapturic acid, and corresponding sulfoxide conjugates of the nephrotoxicant fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether were compared in human proximal tubular cells (Altuntas et al. 2003). Both S-(1,1-difluoro-2-fluoromethoxy-2-[trifluoromethyl]ethyl)-L-cysteine sulfoxide and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-[trifluoromethyl]vinyl)-L-cysteine sulfoxide caused greater cytotoxicity than the corresponding equimolar cysteine conjugates. Toxicity of trichloroethylene S-conjugate sulfoxides occurs via a mechanism independent of β-lyase. Whereas the β-lyase inhibitor aminooxyacetic acid partially protected against S-(1,2-dichlorovinyl)-L-cysteine renal toxicity in vitro and in vivo, it failed to protect against S-(1,2-dichlorovinyl)-L-cysteine sulfoxide toxicity in both settings (Lash et al. 1994). Similarly, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxide toxicities also were not blocked by aminooxyacetic acid (Werner et al. 1996). The α-methyl analog of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide, which is not a substrate for renal β-lyase, also caused renal tubular necrosis in rats in vivo (Birner et al. 1998). S-(1,2-Dichlorovinyl)-L-cysteine sulfoxidation has been proposed as a mechanism to explain the observation that the D- and L- isomers of S-(1,2-dichlorovinyl)-L-cysteine are nearly equally nephrotoxic in rats, yet only the L-isomer is a substrate for β-lyase (Sausen and Elfarra 1991). Thus, both β-lyase-dependent metabolism of cysteine S-conjugates,

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues and CYP450- or flavin monooxygenase-dependent sulfoxidation of cysteine S-conjugates or their mercapturates, can contribute to the bioactivation and renal toxicity of trichloroethylene and other haloalkenes. Several questions remain unaddressed, the answers to which might have important implications for human trichloroethylene biotransformation, toxification, and individual susceptibility. Sulfoxides are more potent nephrotoxicants than their parent S-conjugates. Whereas rat liver microsomes catalyze S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation, and human liver microsomes catalyze S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation, whether human liver microsomes form N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxides remains unknown. The enzymes responsible for human liver (and kidney, if extant) S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation, and particularly the role of CYP3A4 and CYP3A5, remain unknown. Interindividual variability in human S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation remains unknown. CYP3A5 is polymorphic for high expression in Caucasian (30%); Japanese (30%); Chinese (40%); and African American, Southeast Asian, Pacific Islander, and Southwestern American Indian (50%) populations (Hustert et al. 2001; Kuehl et al. 2001; see OMIM 2006a). Assuming that, like rat CYP3A, human CYP3A catalyzes these reactions, together with human CYP3A5 polymorphic expression, suggests that the potential exists for pharmacogenetic differences in sulfoxidation and hence susceptibility to toxicity. This remains unknown, as does the ability of human kidney (which constitutively expresses CYP3A as the major CYP isoform) to catalyze N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation. More fundamentally, the existence of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxidation in vivo has not been documented either in rats or in humans. Myriad investigations of in vivo trichloroethylene disposition in rodents and humans after controlled as well as occupational exposure (Birner et al. 1993; Bernauer et al. 1996; Bruning et al. 1998; Bloemen et al. 2001) were evaluated, including one with 10 metabolites of S-(1,2-dichlorovinyl)-L-cysteine (Bloemen et al. 2001); none evaluated the potential existence of trichloroethylene S-conjugates sulfoxides in urine. Similarly, little is known about sulfoxidation in vivo for any nephrotoxic haloalkene. Only two reports have evaluated sulfoxidation in vivo. In rats, N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide was qualitatively identified in urine after administration of hexachlorobutadiene but was not quantified (Birner et al. 1995). N-Acetyl-S-(1,1-difluoro-2-fluoromethoxy-2-[trifluoromethyl]ethyl)-L-cysteine sulfoxide was identified and quantified in the urine of rats given fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (Sheffels et al. 2004). Although sulfoxidation was apparently a quantitatively small fraction of

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues the overall metabolism, it appeared to be a toxicologically significant route of biotransformation of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether and its S-conjugates. S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide were shown to be formed by rodent liver microsomes (Werner et al. 1996; Ripp et al. 1997) and to be highly reactive renal tubular cell nephrotoxicants in rats in vitro and in vivo (Sausen and Elfarra 1991; Lash et al. 1994, 2003; Rosner and Dekant 1999) and were proposed as important determinants of trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine nephrotoxicity (Krause et al. 2003; Lash et al. 2003); yet no published studies have evaluated S-conjugate sulfoxidation from trichloroethylene or S-(1,2-dichlorovinyl)-L-cysteine, either in rats or in humans, or the toxicologic significance of the conjugates. Such studies may be complicated, however, by the reactivity of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-1,2-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (Sausen and Elfarra 1991; Ripp et al. 1997; Rosner and Dekant 1999), which might therefore not be excreted unchanged. For example, when rats were administered S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, the glutathione conjugate of this sulfoxide was excreted in bile (Sausen and Elfarra 1991; Rosner and Dekant 1999). Therefore, measurement of the glutathione conjugate of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide or its metabolites may provide a method to assess S-(1,2-dichlorovinyl)-L-cysteine sulfoxide formation after trichloroethylene or S-(1,2-dichlorovinyl)-L-cysteine exposure in vivo. NONCANCER TOXICITY Animal Studies Tubular Toxicity Trichloroethylene has been shown to cause toxicity to renal tubules in bioassay studies, and mechanisms of this toxicity have been pursued in experimental studies. Lash et al. (2000b) reviewed mechanistic studies and those will not be recapitulated here. The committee directed its efforts to studies since that review. Trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine are toxic to primary cultures of rat proximal and distal tubular cells (Cummings et al. 2000). Glutathione-related enzyme activities were well maintained in the cells, whereas CYP activities were not. The response to S-(1,2-dichlorovinyl)-L-cysteine was greater than the response to trichloroethylene; however, the proximal and distal tubule cells had similar responses even though the proximal tubule is the target in vivo. The authors attributed this to the fact that the proximal tubule is exposed before the distal tubule in vivo and to

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues possible differences in uptake transporters. They did not address the extent to which transporters were maintained in the cultured cells. The same group also assessed the toxicity of trichloroethylene and its metabolites S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione using in vitro techniques (Lash et al. 2001b). Their goal was to determine whether in vitro techniques are valid indicators of species-, sex-, and tissue-related differences in sensitivity. Experiments using isolated cells were performed only with tissues from Fischer 344 rats, and lactate dehydrogenase release was used as the measure of cellular toxicity. The effects were greater in males. S-(1,2-Dichlorovinyl)-L-cysteine and trichloroethylene had similar effects, but S-(1,2-dichlorovinyl)glutathione exhibited increased efficacy compared with trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine. Mitochondrial toxicity was assessed in both Fischer 344 rats and B6C3F1 mice. Renal mitochondria from male rats and mice responded similarly; a greater effect was seen in female mice. Thus, although these studies show S-(1,2-dichlorovinyl)-L-cysteine to be more toxic than trichloroethylene and S-(1,2-dichlorovinyl)glutathione, the magnitude of the effect was not much different and species differences are not consistent with the effects observed in long-term bioassays. This suggests that in vitro data be used with caution in risk assessment, being mindful that in vitro experiments avoid in vivo pharmacokinetic and metabolic processes. In LLC-PK1 cells, S-(1,2-dichlorovinyl)-L-cysteine causes loss of mitochondrial membrane potential, mitochondrial swelling, release of cytochrome c, caspase activation, and apoptosis (Chen et al. 2001). Thus, S-(1,2-dichlorovinyl)-L-cysteine is toxic to mitochondria, resulting in either apoptosis or necrosis. S-(1,2-Dichlorovinyl)-L-cysteine-induced apoptosis also has been reported in primary cultures of human proximal tubule cells (Lash et al. 2001a). Korrapati et al. (2005) builds upon a series of investigations of hetero-(by HgCl2) and homo-(by S-(1,2-dichlorovinyl)-L-cysteine, 15 mg/kg) protection against a lethal dose of S-(1,2-dichlorovinyl)-L-cysteine (75 mg/kg), in which priming, or preconditioning, was said to augment and sustain cell division and tissue repair, hence protecting against the subsequent lethal S-(1,2-dichlorovinyl)-L-cysteine dose (Vaidya et al. 2003a,b,c). Korrapati et al. (2005) showed that a lethal dose of S-(1,2-dichlorovinyl)-L-cysteine downregulates phosphorylation of endogenous retinoblastoma protein (pRb), which is considered critical in renal proximal tubular and mesangial cells for the passage of cells from G1 to S-phase, thereby leading to a block of renal tubule repair. Priming, in contrast, upregulated P-pRb which was sustained even after the administration of a lethal dose of S-(1,2-dichlorovinyl)-L-cysteine, thereby stimulating S-phase DNA synthesis, which was concluded to result in tissue repair and recovery from acute renal failure

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues and death. While these studies are indeed fascinating, they inform more on the mechanism of autoprotection rather than on the mechanism of initial injury caused by S-(1,2-dichlorovinyl)-L-cysteine. In addition, the priming injury (not innocuous, as it caused 25-50% necrosis and elevated blood urea nitrogen) may have influenced the toxicokinetics of the second S-(1,2-dichlorovinyl)-L-cysteine injection. This remains unknown. Mensing et al. (2002) reported on the nephrotoxicity of trichloroethylene in male Long Evans rats after 6 months of inhalation exposure (500 ppm). Results were expressed relative to urine creatinine to account for individual differences in urine volume that can affect the concentration of urine constituents. Urinary excretion of albumin was not affected (although the high end of the range was about twice that of the control group) and high-molecular-weight proteins showed an upward trend but were not significantly increased (creatinine at 36 mg/g [4-81 mg/g] versus 41 mg/g [not detected-215 mg/g]). Increased excretion of low-molecular-weight proteins and N-acetylglucosaminidase was noted. The increase in N-acetylglucosaminidase was small (8.4 units [U]/g [5.7-8.9 U/g] versus 9.7 U/g [not detected-12.4 U/g); the increase in low-molecular-weight proteins was 332 U/g (176-659 U/g) versus 637 U/g (293-1,910 U/g). The histopathology description does not mention tubular damage, whereas interstitial infections and glomerulonephritis are described for the treated group. Proteinuria has long been recognized as a sign of kidney damage, and it is a reliable predictor of ultimate outcome; more recently, it has been recognized that an elevated filtered load of protein is damaging to tubules (Verhave et al. 2004; Zandi-Nejad et al. 2004). Proteinuria can be characterized as glomerular, tubular, or mixed, based on the causal defect. Proteins less than about 40 kDa or 30 Å are readily filtered at the glomerulus, and are reabsorbed in the proximal tubule. Proteins larger than 100 kDa or 55 Å are not filtered. Albumin is considered an intermediate-sized protein that is normally filtered sparingly, largely because of its negative charge being repulsed by a fixed negative charge in the glomerular barrier. The glomerular pattern is excretion of high-molecular-weight proteins, such as IgG, and indicates increased permeability or decreased selectivity of the glomerular barrier. Damage to the proximal tubule impairs reabsorption of low-molecular-weight proteins; thus, a tubular pattern is one that has increased excretion of albumin and low-molecular-weight proteins, such as α1-microglobulin. N-Acetylglucosaminidase is a lysosomal protein released by tubules during processing of filtered protein. Increased amounts of N-acetylglucosaminidase are expected when the tubules are presented with elevated amounts of protein, and thus it is an indicator of protein load. Elevated urinary N-acetylglucosaminidase is not an index of cell death, as is release of alkaline phosphatase from cultured cells or release of trans-

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues aminase enzymes from liver cells into the plasma. However, elevation of N-acetylglucosaminidase in urine is a sign of proteinuria, which is a sign of kidney malfunction (Zandi-Nejad et al. 2004). While Mensing et al. (2002) did not report tubular toxicity, the urinary protein profile is consistent with impairment of tubule reabsorption of filtered protein and perhaps increased glomerular permeability to proteins. Role of Formic Acid in Trichloroethylene Nephrotoxicity Some investigators (Green et al. 1998, 2003; Dow and Green 2000) have proposed that the mode of trichloroethylene nephrotoxicity is related to formic acid. They demonstrated that exposure to either trichloroethanol or trichloroacetic acid causes increased formation and urinary excretion of formic acid (Green et al. 1998). The formic acid does not come from trichloroethylene (Figure 3-1). Rather, trichloroethylene (or a metabolite) causes a functional depletion of vitamin B12, which is required for the methionine salvage pathway of folate metabolism. Vitamin B12 depletion results in folate depletion. Folate is a cofactor in one-carbon metabolism and depletion of folate allows formic acid to accumulate, and then to be excreted in the urine (Dow and Green 2000). The effects of trichloroethanol-induced formic acid accumulation were determined in a 1-year chronic toxicity study in male Fischer 344 rats (Green et al. 2003). Trichloroethanol was administered in drinking water to achieve a urine formic acid concentration similar to that found in rats exposed by inhalation to trichloroethylene at 500 parts per million (ppm). The pathology of formic acid (induced by trichloroethanol administration) is initially increased tubular basophilia and hyaline drop accumulation (12-16 weeks) followed by tubular degeneration at 40 weeks (“increased cellular eosinophilia, tubular vacuolation and intratubular cast formation”) and an increased amount of pigmentation in the S2 portion of the proximal tubules and hyaline droplet accumulation. At 52 weeks, hyaline droplet and tubular degeneration were not found, but increased tubular pigmentation was observed. It was also noted that foci of “atypical” tubular hyperplasia occurred in two of the trichloroethanol-treated rats. The authors stated these changes were consistent with the nephrotoxicity seen in the 2-year cancer bioassays. Results from the National Toxicology Program’s 2-year cancer bioassays of trichloroethylene administered by gavage to rats and mice are provided in Tables 3-1 and 3-2. Nonneoplastic kidney lesions were found in all animals dosed for 2 years, including mice that did not develop kidney cancer (NTP 1988). In rats, both studies noted cytomegaly and karyomegaly of tubular cells in the area of the corticomedullary border (specified as pars recta by NTP [1990], which is situated in the corticomedullary region).

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues this “case-only” analysis is that it does not require use of the control series (analogous to a case-only study in gene-environment interactions); it was unclear whether there was blinding of exposure status when the molecular analyses were conducted. Role of Metabolism in Trichloroethylene-Induced Renal Tumors Extensive studies of trichloroethylene metabolism, coupled to its potential mechanism of action in nephrocarcinogenicity, have been reported (reviewed by Brüning and Bolt 2000). Trichloroethylene induces renal toxicity and renal tumors in rats (Maltoni et al. 1988; NTP 1988, 1990; EPA 2001). The nephrocarcinogenic effects of trichloroethylene are more pronounced in male rats, compared with female rats and were absent in male and female mice.2 Studies of trichloroethylene metabolism in rodents and humans support a role for bioactivation in the development of nephrotoxicity and nephrocarcinogenicity after exposure to trichloroethylene (Lash et al. 1995, 2001a,b, 2002, 2003; Lash 2004). Trichloroethylene is metabolized by two competing pathways: oxidation by CYP450 and conjugation with glutathione (discussed earlier in this chapter; see Figure 3-1). Glutathione conjugation of trichloroethylene results in formation of S-(dichlorovinyl)glutathione, which is metabolized by enzymes of the mercapturic acid pathway (γ-glutamyl transpeptidase, aminopeptidase) to S-(1,2-dichlorovinyl)-L-cysteine, which is then metabolized by cysteine conjugate β-lyases, leading to the formation of electrophilic chlorothioketenes and sulfoxides. Concentrations of trichloroethylene in renal cortical homogenates have been reported to be generally two- to three-fold higher than in liver homogenates, and both oxidative and glutathione conjugation products were found in the liver and kidneys (Lash et al. 2006). These results are consistent with in vitro studies showing metabolism by kidney tissue. Males had substantially higher urinary excretion of S-(1,2-dichlorovinyl)-L-cysteine, suggesting greater metabolism by the glutathione pathway. It should be noted that results were reported for only three animals per time point and interpretation of the data is complicated by anomalous dose-concentration time profiles for trichloroethylene and its metabolites. The nephrotoxicity and nephrocarcinogenicity of trichloroethylene have been linked to the formation of S-(1,2-dichlorovinyl)-L-cysteine derivatives. 2 Trichloroethylene is described in the literature as being carcinogenic in males only. The magnitude of effect is smaller in females and did not reach statistical significance in the individual studies. EPA (2001b) did an analysis of modified data. The results across strains were pooled and animals that died before any tumors were observed were removed from the analysis. With these modifications, the tumor effect in females was significant.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues S-(1,2-Dichlorovinyl)-L-cysteine and its mercapturic acid metabolite N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine have been identified in the urine of humans exposed to trichloroethylene, providing evidence for the glutathione-dependent bioactivation of trichloroethylene in humans. Metabolism of trichloroethylene via the mercapturic acid metabolic pathway is consistent with the fact that the male rat is a sensitive species, because reduced glutathione (GSH) conjugation, γ-glutamyl transpeptidase, and cysteine conjugate β-lyase activity are all significantly higher in male than in female rats (Lash et al. 2002). Moreover, pharmacokinetic analysis of human volunteers after exposure to trichloroethylene (50 or 100 ppm) revealed that blood S-(dichlorovinyl)glutathione concentrations were 3.4-fold higher in males than in females, whereas clearance half-time values for systemic clearance of S-(dichlorovinyl)glutathione were similar in both genders (Lash et al. 1999). In the liver, metabolism of trichloroethylene via the mercapturic acid metabolic pathway is quantitatively less than via the CYP450-dependent metabolic pathway. However, the glutathione-dependent pathway becomes more pronounced when the oxidative metabolism of trichloroethylene is saturated in the case of high-dose exposure. Cummings and Lash (2000) demonstrated that human kidney tissue forms GSH conjugates with a Km (0.58 mM) in the range of Km values for oxidative metabolism by rodent microsomes (0.38 mM for mice, 0.07 and 0.48 mM for rats; Table C-2). They reported minimal or nondetectable P450-mediated trichloroethylene metabolism in human kidney tissue. Genotoxicity Trichloroethylene causes a significant increase in the incidence of renal tumors in rats when administered orally and a marginal incidence of renal tumors when administered via inhalation; on the basis of limited evidence for carcinogenicity in humans and sufficient evidence for carcinogenicity in experimental animals, the International Agency for Research on Cancer (IARC 1995b) classified trichloroethylene as a probable carcinogen in humans (group 2A). Moore and Harrington-Brock (2000) reviewed the genotoxicity of trichloroethylene and its glutathione-derived metabolites, and Brüning and Bolt (2000) reviewed the results of genotoxicity tests and concluded that trichloroethylene is, at most, a weak genotoxicant but noted that S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine have genotoxic effects including mutagenicity in the Ames test, unscheduled DNA synthesis, and formation of adducts in vitro with adenine, cytosine, and guanine. In the preliminary screening phase, the standard battery of genotoxicity tests might be unable to identify tissue-specific carcinogens, if the test system lacks the enzymes needed to form the toxic metabolite, and

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues certainly does not provide any information on the possible species specificity of the test compound (Brambilla and Martelli 2004; Moore and Harrington-Brock 2000). Recently, Robbiano et al. (2004) applied both in vitro and in vivo assays to measure genotoxicity to kidneys of rodents and repeated the assays in primary cultures of human kidney cells. Six chemicals known to induce kidney tumors in rats, including trichloroethylene, were examined for their ability to induce DNA fragmentation and the formation of micronuclei in primary cultures of rat and human kidney cells and in kidneys of intact rats. Each chemical was tested at three to six concentrations (four 2-fold dilutions for trichloroethylene); the highest concentration tested produced a less than 30% reduction in survival. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites (as measured by the Comet assay) and in micronuclei frequency were obtained in primary kidney cells from male rats and from humans of both genders, with subtoxic concentrations of trichloroethylene. Among the six test compounds (benzofuran, bromodichloromethane, captafol, nitrobenzene, ochratoxin A, and trichloroethylene), trichloroethylene and bromodichloromethane exhibited the lowest DNA-damaging and micronuclei-inducing potencies (with ochratoxin A exhibiting the highest) in rats and humans. In agreement with these findings, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were observed in the kidneys of rats given a single oral dose (half the lethal dose to 50% of rats) of the six test compounds. For all these effects, the magnitude of the response was among the greatest for trichloroethylene. The results of this study also showed that the six rat kidney carcinogens produced genotoxic effects in primary cultures of human kidney cells that were quantitatively and qualitatively similar to those observed in primary cultures of rat kidney cells. Taken together, these findings provide evidence that trichloroethylene is genotoxic in short-term genotoxicity assays in kidney cells isolated from rats and human donors. However, the authors noted limitations in the experimental design that limit interpretation and the significance of the above studies (Robbiano et al. 2004). These limitations include (1) examination of trichloroethylene on cells from only three donors, (2) considerable variation in the frequency of DNA lesions induced in the cells, and (3) the possibility that kidney cells derived from kidney cancer patients could be more sensitive to DNA-damaging activity due to a more marked expression of enzymes involved in the metabolic activation of kidney procarcinogens and suppression of DNA repair processes. Therefore, the results of the genotoxicity studies must be considered solely as indicating that trichloroethylene might be genotoxic to the human kidney; the authors suggest that the designation of “inadequate evidence for carcinogenicity to humans” might not be tenable in the absence of sufficiently powered and carefully controlled epidemiologic studies.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Mode of Action Role of von Hippel-Lindau Tumor Suppressor Gene Most of the studies thus far reviewed on the subject of the renal carcinogenicity of trichloroethylene rely on either epidemiologic approaches or on studies of trichloroethylene metabolism and toxicity. The development of DNA technology and the discovery of tumor suppressor genes opened up a new route of investigation on the potential carcinogenic effects of trichloroethylene. Mutation or inactivation of the p53 tumor suppressor gene is a common genetic alteration in human cancers. However, the p53 gene is not a target in human and rat renal cell carcinoma (Reiter et al. 1993; Nishiyama et al. 1995). Inactivation of the VHL tumor suppressor gene in humans is responsible for the hereditary VHL cancer syndrome, predisposing affected individuals to a variety of tumors in specific target organs. More than 80% of sporadic renal cell carcinoma, but not papillary renal cell carcinoma, is associated with inactivation of the VHL gene (Gnarra et al. 1994). The VHL gene is only infrequently involved in extrarenal neoplasms, despite the broad range of VHL mRNA expression (including brain, adrenal, prostate, and lung), suggesting that its function as a tumor suppressor gene is specific for kidney epithelial cells (Walker 1998). The protein product of the VHL gene appears to regulate cell cycle arrest (transition from G1 to G0) by stabilizing the cyclin-dependent kinase inhibitor p27 (Soucek et al. 1998). Although the VHL gene, which is commonly mutated in human renal cell carcinoma, does not appear to be involved in rat renal cell carcinoma (Walker et al. 1996), it shares a common downstream effector (p27 that controls cell cycle progression) with the TSC2 gene, a genetic target of renal cell carcinoma development in the rat. Because VHL is not a target gene in rodent models of chemical-induced or spontaneous renal carcinogenesis, future animal studies should use models in which target genes share common downstream signaling pathways with VHL. One paper has linked the VHL gene to chemical-induced carcinogenesis. Shiao et al. (1998) demonstrated VHL gene somatic mutations in N-nitrosodimethylamine-induced rat kidney cancers that were of the clear cell type. The clear cell phenotype is rare in rat kidney cancers, but it was the only the clear cell cancers that showed VHL somatic mutation. This provided an additional link between VHL inactivation and clear cell kidney cancer. Brauch et al. (1999, 2004) analyzed renal cancer cell tissues for mutations of the VHL gene and reported increased occurrence of mutations in patients exposed to high concentrations of trichloroethylene. In the first study (Brauch et al. 1999), subjects were identified from an occupational trichloroethylene exposure registry. They found multiple mutations in 42%

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues of the exposed patients who experienced any mutation and 57% showed loss of heterozygosity. A hot spot mutation of cytosine to thymine at nucleotide 454 (C454T) was found in 39% of samples that had a VHL mutation and was not found in renal cell cancers from nonexposed patients or in lymphocyte DNA from either exposed or nonexposed cases or controls. As discussed earlier, little information was given on how subjects were selected and whether there was blinding of exposure status during the DNA analysis. In the second study, Brauch et al. (2004) investigated 38 renal cell carcinoma patients from a previous German case-control study performed by Vamvakas et al. (1998). Brauch et al. compared different renal cell carcinoma patient groups (trichloroethylene-exposed versus nontrichloroethylene-exposed patients). The Vamvakas et al. study had described differences in renal cell carcinoma risks between trichloroethylene-exposed (n = 17) and nonexposed patients (n = 21). Brauch et al. (2004) extended the analysis by comparing age at diagnosis and histopathologic parameters of tumors as well as somatic mutation characteristics in the VHL tumor suppressor gene. Renal cell carcinoma did not differ with respect to histopathologic characteristics in both patient groups. Comparing results from trichloroethylene-exposed and nonexposed patients revealed clear differences with respect to (1) frequency of somatic VHL mutations, (2) incidence of C454T transition, and (3) incidence of multiple mutations. The latter is an indication that the effect of trichloroethylene is not limited to clonal expansion of cells mutated by some other agent. The C454T hot spot mutation was exclusively detected in tumors from trichloroethylene-exposed patients, as were multiple mutations. Also the incidence of VHL mutations in the trichloroethylene-exposed group was at least 2-fold higher than in the nonexposed group. Brauch et al. were not able to analyze all the samples from the Vamvakas study, in part because samples were no longer available. Using the data described by Brauch et al. (2004) (VHL mutation found in 15 exposed and 2 nonexposed individuals, and VHL mutation not found in 2 exposed and 19 unexposed individuals), an OR of 71.3 is calculated. The most extreme example would be to assume that all 20 cases who were excluded were exposed but did not have mutations in VHL (VHL mutations were found in 15 exposed and 2 unexposed individuals and VHL was not found in 22 exposed and 18 unexposed individuals), which leads to an OR of 6.5, which remains significant. Collectively, the data support the concept of a genotoxic effect of trichloroethylene leading to VHL gene damage and subsequent occurrence of renal cell carcinoma in highly exposed subjects. All the evidence, taken together, provides a consistent and plausible mechanism for a causal relationship and is strongly supportive of trichloroethylene being a human carcinogen after long-term exposure to high doses, such as occupational

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues exposures described in both studies conducted in Germany (Henschler et al. 1995; Vamvakas et al. 1998). The VHL gene is commonly altered in kidney tumors, especially those with the clear cell phenotypes. The alterations include loss of the entire or a large part of the gene (>90%) and small base changes (30% to 60%), including insertions, deletions, and point mutations (Shiao 2004). These changes can lead to reduction of protein expression, protein truncation, and incorrect amino acids incorporated into the protein (also called missense mutation). Consequently, wild-type constitutive functions of VHL are inactivated, with the subsequent potential to initiate and to promote tumor development. However, different mutations might have distinct tumorigenic potentials. Frequent and diverse VHL mutations in sporadic renal cell carcinoma provide a sizable mutation spectrum that has been used to correlate with environmental exposures. The rationale of using genetic signature as a marker of environmental exposure has been strengthened by in vitro and in vivo studies. Correlating of specific mutations within the VHL gene with certain environmental exposures could lend support to the potential mutagenicity of an agent. Identification of DNA damage unique to exposure is necessary to provide strong evidence for the mutagenic potential of an environmental agent. Many types of DNA damage have been shown to induce unique signatures of gene mutations (see Table 3-14). A worldwide mutation database compiling VHL mutations in sporadic renal cell carcinoma showed that missense mutations compose about 29% of all mutations; a large majority of base changes (71%) are nonmissense, including insertions, deletions, and frameshift alterations (see Table 3-15). When bases were determined, G:C to A:T, A:T to G:C, and A:T to C:G composed 48% of the changes. Similar mutation spectra have been obtained TABLE 3-14 Mutation Spectra Indicative of Environmental Exposures and DNA Damage Base Change Possible Causes Transition   G:C to A:T Deamination of 5-methyl-C or C; alkylation of G at O6 position A:T to G:C Deamination of A; alkylation of T at O2 or O4 position Transversion   G:C to T:A Mispairing of A with 8-OH-G or with apurinic G A:T to T:A Mispairing of A with apurinic A site A:T to C:G Misincorporation of 8-OH-G; error-prone repair of O2- or O4-alkyl T G:C to C:G Mispairing of G with oxidatively damaged G SOURCE: Shiao 2004. Reprinted with permission; copyright 2004, National Cancer Institute at Frederick.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TABLE 3-15 VHL Mutations in Sporadic Renal Cell Carcinomas   Trichloroethylene Exposure Brüning et al. 1997a Brauch et al. 1999 Brauch et al. 2004 UMDa Yes Yes No Yes No Unknown Number of patients 23 44 73 17 21   Patients with mutations 23 (100%) 33 (75%) 42 (58%) 14 (82%) 2 (10%)   Number of mutations 23b 50 42 24 2 222 Missense 1 27 (54%) NA 17 (71%) 2 (100%) 64 (29%) Nonmissense 3 23 (46%) NA 7 (29%) 0 (0%) 158 (71%) G:C to A:T 1 21 (78%) NA 12 (71%) 1 (50%) 21 (25%) C to T at 454   (13) (0/107) (9) (0) (0) G:C to T:A   0 NA 0 0 19 (22%) G:C to C:G   5 (19%) NA 4 (24%) 0 16 (19%) A:T to T:A   1 (4%) NA 1 (6%) 0 9 (11%) A:T to G:C   0 NA 0 1(50%) 14 (16%) A:T to C:G   0 NA 0 0 6 (7%) aUniversal Mutation Database (Beroud et al. 2000). bBy single-strand conformation polymorphism (4 sequences confirmed). ABBREVIATION: NA, not applicable. from cells and animals treated with alkylating agents, such as nitrosamines found in tobacco smoke and potent human and animal renal carcinogens. The involvement of alkylating agents in the causation of renal cell carcinoma is further supported by the isolation of O6-methylguanine and other alkylated DNA-damaged bases. However, mutation spectra after exposure to trichloroethylene or analog compounds, in cells and animals, have not been consistent. Nonetheless, increases in GC to AT and GC to TA mutations have been observed in bacteria. Muller et al. (1998) identified cytosine adducts from haloketene and halothioketene products of trichloroethylene; these are structurally similar to hydroxylamine cytosine adducts that result in C to T mutations (Budowsky 1976). Increases in VHL missense mutations, predominant in G:C to A:T base changes, and a hot spot of mutation at nucleotide 454, correlated with trichloroethylene exposure (Brauch et al. 1999). The three reports of trichloroethylene exposure from the same group suggest that trichloroethylene increases VHL mutations and generates a unique genetic signature of trichloroethylene exposure, which leads to the development of renal cell carcinoma. Although the findings linking trichloroethylene to renal cancer are of great consequence and relevance, further

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues confirmation of mutagenicity and carcinogenicity at the molecular level is required to confirm the initial observations. As discussed earlier, consensus for the mutagenicity of trichloroethylene in mammalian cells remains to be established. If the mutation spectra in bacteria are considered, one would expect to see increases of both G:C to A:T and G:C to T:A mutations in trichloroethylene-exposed humans. However, a disproportionate number of G:C to A:T VHL mutations were reported (Brauch et al. 1999). Because alkylating agents, present in patients exposed to tobacco smoke, diuretic treatment for hypertension, and long-term dialysis for end-stage renal failure, also induce the same G:C to A:T base changes, analysis of prior trichloroethylene studies need to adjust for these risk factors. The temporal relationship between various mutations in the VHL gene and renal tumor progression needs to be examined more critically to unequivocally evaluate the cause-and-effect relationships. The mutagenicity of trichloroethylene should also be validated in additional cohorts. Further, the tumorigenic potentials of various VHL mutations need to be integrated, because mutated bases need not always be carcinogenic. It remains debatable whether alterations in VHL alone are sufficient to trigger tumorigenic processes in the kidney, especially since experiments failed to detect any tumors in VHL knockout mice (Gnarra et al. 1997; Haase et al. 2001). Studies attempting to link the VHL gene to kidney tumor development are continuing in a variety of experimental models (Shiao et al. 1997, 1998; Walker 1998). However, there does not appear to be an experimental animal model with which to investigate the effects of trichloroethylene-induced mutations in the VHL gene and kidney tumor development. Role of Nephrotoxicity in Trichloroethylene Renal Cancer In animal studies, renal cancer occurs at high doses and is preceded by nephrotoxicity affecting the proximal tubule (NTP 1988, 1990). This has led to the proposal that nephrotoxicity is a prerequisite for the development of renal tumors and that exposures below nephrotoxic concentrations pose no risk of cancer. That is, there is a threshold exposure below which nephrotoxicity, and therefore renal cancer, will not occur (Brüning and Bolt 2000; Harth et al. 2005). In this scenario, nephrotoxicity, and subsequent cell division repairing that damage, functions as a promoter, allowing the expression of mutations (either spontaneous or induced by exposure to other agents, such as smoking and diuretics) within the renal cortex. Alternatively, trichloroethylene is a complete carcinogen, with nephrotoxicity as the promoter for cells initiated by a trichloroethylene metabolite. There is evidence that trichloroethylene is genotoxic to human cells (Robbiano et al. 2004).

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Nephrotoxicity is almost certainly secondary to formation of a toxic metabolite, and species differences in the extent of formation of that toxic metabolite could render humans less likely to develop nephrotoxicity and therefore cancer. The CYP2E1 and -3A5 isoforms that metabolize trichloroethylene have polymorphisms within national populations, resulting in considerable interindividual differences of enzyme expression. On a practical level, the population diversity in bioactivation and detoxification abilities could effectively obscure any threshold. Investigations of nephrotoxicity in human populations have been pursued and the results show that highly exposed workers experience a tubular type of proteinuria, evidence of damage to the proximal tubule (Brüning et al. 1999a,b; Bolt et al. 2004). What is not clear is the magnitude of exposure needed to produce kidney damage. The fact that proteinuria was found in workers exposed to trichloroethylene concentrations that were not measured but were described as current occupational exposures (Green et al. 2004) is inconsistent with nephrotoxicity occurring only at high exposures that are not relevant to current occupational exposures. FINDINGS Although the committee was not charged with performing a risk assessment, it became clear from the epidemiologic evidence that there were sufficient data to make a recommendation about whether the findings of the mortality and incidence studies provided support for or against the hypothesis that exposure to trichloroethylene was associated with the induction of kidney cancer. There is strong evidence that exposure to high doses of trichloroethylene is associated with increased rates of kidney cancer. In particular, support for this conclusion derives from findings of increased risks in a cohort study (Henschler et al. 1995) and in case-control studies from the Arnsburg region of Germany (Vamvakas et al. 1998; Pesch et al. 2000a; Brüning et al. 2003). The committee notes that, as the designs of these case-control studies improved with time, increased risks were still observed. In addition, the finding of a mutation in the VHL somatic gene adds strength to these observations, although it would be useful if this finding were replicated in other settings. Of considerable interest was the finding of an increased risk among workers of a cardboard manufacturing plant in the United States (Sinks et al. 1992), who might have had exposures comparable to that in the study by Henschler et al. (1995). Other studies with appropriate power to detect risks from relatively low exposures also showed increased risks, notably the studies by Dosemeci et al. (1999), Raaschou-Nielsen et al. (2003), and Zhao et al. (2005). Supporting this conclusion is the concordance between studies on humans and experimental animals for the site of tumors and occurrence of

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues toxicity. In bioassay studies, rats developed tubular toxicity before tumors developed. Nephrotoxicity preceding cancer also appears likely in humans, although nephrotoxicity assessments in human studies were not made until after the development of renal cancer and were based on only one parameter. The committee reviewed studies on two modes of toxicity proposed to be linked to cancer—accumulation of α2µ-globulin and PPAR agonism. The committee concluded the evidence demonstrates these modes do not occur for trichloroethylene-induced renal cancer. The committee also concluded that trichloroethylene causes an increase in the urinary excretion of formate but notes the disparities between formate production and toxicity contradicts the conclusion that accumulation of formate is a mode of action for trichloroethylene nephrotoxicity. Studies with experimental animals and human tissues support the conclusion that trichloroethylene, via one or more of its metabolites, is genotoxic. In animal studies, trichloroethylene appears to be a weak genotoxicant. The studies with human tissues used a small number of samples and, therefore, the committee notes this weakens the weight of evidence. In the kidney, trichloroethylene can act as a complete carcinogen (at the stages of both tumor initiation and tumor promotion and progression) in a dose-dependent manner. Different types of kidney cancer can be triggered by different genes. After the discovery of the VHL tumor suppressor gene, it became recognized that homozygous inactivation of the VHL gene was linked to the occurrence of renal clear cell carcinoma, the renal carcinoma preferentially induced by trichloroethylene. In exposed subjects, the genotoxic effect of trichloroethylene likely results from bioactivation pathways leading to renal VHL gene damage and renal cell carcinomas. The findings of experimental, mechanistic, and epidemiologic studies lead to the conclusion that trichloroethylene can be considered a potential human carcinogen. RESEARCH RECOMMENDATIONS Because sulfoxide metabolites are more potent nephrotoxicants than their parent S-conjugates, more research is needed on the extent of formation of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxides by human tissues (liver and kidney), the extent to which these reactions occur in vivo, the enzymes involved, and their interindividual variability, including the role of genetic polymorphisms. The toxicologic significance of trichloroethylene or S-(1,2-dichlorovinyl)-L-cysteine S-conjugate sulfoxidation products also should be evaluated. High frequencies of missense mutations in the VHL gene do not constitute a cause of renal cell carcinoma; the tumorigenic potential of missense

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues mutations in the VHL gene should be determined. The potential of specific missense mutations in the VHL gene contributing to tumor initiation and progression should be determined. Although correlation of VHL mutations to trichloroethylene exposure and renal cell cancer are persuasive, the findings need to be validated in other populations and geographic areas. Because many risk factors for renal cell carcinoma generate mutation spectra similar to that of trichloroethylene, coexposure to trichloroethylene with other risk factors needs to be seriously considered and accounted for in future epidemiologic studies. Mechanistic studies should include field studies of populations exposed to trichloroethylene to assess the range of metabolic pathways used and relative amounts of metabolites from each pathway as a function of exposure intensity and enzymatic genotypes. This information will greatly help in the interpretation and extrapolation of information from rodents to humans. Additional studies of nephrotoxicity in workers exposed occupationally to trichloroethylene should be performed. It is important that actual exposures are measured and not estimated using biological markers that are subject to large interindividual differences. No analytic community studies were included in the committee’s assessment of kidney cancer. Given the importance of contamination of water supplies by trichloroethylene, it is important that sufficiently robust studies (with sufficient statistical power and exposure assessments) be conducted in the general population where such exposures might be occurring. Any follow-up epidemiologic study must have a wide range of exposures, preferably to the range of the Vamvakas and Henschler studies to provide an anchor in that range where effects were seen. There may be opportunities for studies of populations in developing countries in Asia and Eastern Europe, where high exposures to trichloroethylene may not have been controlled. Strong, quantitative exposure assessments will be critical for these studies to be useful for resolving the remaining dose-response issues.