I. Introduction

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Perchloroethylene (Perc²; also known as tetrachloroethylene or tetrachloroethene) is a widely used dry cleaning and metal degreasing solvent. It is a hazardous air pollutant, a common contaminant at Superfund waste sites, and is a surface and ground water pollutant. Over 400 million pounds of Perc are produced annually in the United States. The primary routes of potential human exposure to Perc are inhalation and dermal contact. Approximately 85% of Perc that is used annually is lost to the atmosphere, so that concentrations in air have been reported to range from 30 ppt in rural areas to as high as 4.5 ppb in urban or industrial areas [National Toxicology Program (NTP), 2001]. Perhaps the greatest concern for the general population is contamination of the drinking water with Perc. Current regulations by the U.S. EPA have established a maximum contaminant level of 0.005 mg of Perc/liter (i.e., 5 ppb) in the drinking water (U.S. EPA, 1989).

Perc has been clearly identified as a carcinogen in experimental animals [International Agency for Research on Cancer (IARC), 1979, 1987; U.S. EPA, 1985, 1991; NTP, 1986] and is considered by the IARC to be probably carcinogenic to humans (group 2A) (IARC, 1995). This evaluation was based on the findings of limited evidence in humans and sufficient evidence in experimental animals of carcinogenicity. IARC also concluded that there is limited evidence in humans for the carcinogenicity of occupational exposures in dry cleaning. Perc is the predominant solvent used in dry cleaning in most areas of the world, including the United States.

The toxic effects of Perc in liver and kidney, which have been observed primarily in experimental animals, are considered to be dependent on its metabolism to reactive metabolites. The pathways for Perc bioactivation and detoxification are rather complex, and several of the enzymes involved exhibit sex- and species-dependent differences. Consequently, it is often difficult to extrapolate results from experimental animals to humans with certainty. Because there is target organ concordance across species for toxicity in general, but not for carcinogenicity, a more complete understanding of the metabolism of Perc in the various target organs and in different species, including humans, is needed to improve predictions for human health risk assessment.

Recent reviews of Perc metabolism and mode of action have been published in a monograph by IARC (1995) and by the U.S. EPA (1991). The IARC monograph contains a concise overview of exposure data, Perc production and use, occupational and environmental occurrence data, and summaries of studies of human cancer that can be attributed to Perc exposure, studies of cancer in experimental animals, Perc metabolism, and target organ toxicity. The EPA document was a response to issues that were raised or left unresolved in the most recent health assessment for Perc (U.S. EPA, 1985) and the addendum that followed (U.S. EPA, 1986). The states of California (California Environmental Protection Agency, 2000) and New York (New York State Department of Health, 1997) and the Agency for Toxic Substances and Disease Registry (ATSDR, 1997) have also conducted health assessment reviews of Perc.

This review will focus on Perc metabolism and modes of action for hepatic and renal toxicities. The first section will outline the two principal pathways of Perc metabolism that occur in the liver and kidney, focusing on identification of metabolites that are critical for toxicity and on tissue-, sex-, and species-dependent differences in flux through the various steps. These two pathways are cytochrome P450 (P450)-dependent oxidation and glutathione (GSH) conjugation. The relationship between these two pathways and between metabolism of Perc and that of trichloroethylene (TRI) will also be considered. Physiologically based pharmacokinetic (PBPK) models have become important tools in the evaluation of experimental data and in extrapolation of animal data to humans. PBPK models that have been developed for Perc will be discussed with a focus on their applicability to human health risk assessment. The next two sections will summarize in vivo studies in laboratory animals and occupational and epidemiological studies in humans. The subsequent two sections consider mechanistic data and proposed modes of action for Perc and some of its metabolites in two target organs, the liver and the kidneys. The scientific plausibility and relevance of the mechanistic information and these proposed modes of action for humans will be evaluated. Finally, a reference dose (RfD) and reference concentration (RfC) for Perc exposure will be developed.

Although TRI and Perc have very similar chemical properties, are often used in industry for the same or similar purposes, are often found together as environmental contaminants, are metabolized by essentially all the same enzymes, share many of the same or similar metabolites, and elicit many of the same toxic effects (Green, 1990; IARC, 1995), it would be a serious mistake to assume a priori that risk of hazard from exposure to the two chemicals is the same or that the modes of action and rates of metabolism in target tissues are identical. This is because significant differences are known in the kinetics of metabolism of TRI and Perc by certain enzymes and in the chemical reactivity of certain analogous metabolites. Much more work has been pursued on the metabolism and mode of action of TRI and its metabolites than for Perc and its metabolites. With the caution mentioned above in mind, reference will be made to studies on TRI or its relevant metabolites where appropriate and with proper qualifications. In some cases, the only potentially relevant information that is available comes from such studies on the Perc congener, TRI, or their common metabolites. This review will not repeat detailed discussion of all the studies presented in earlier documents (IARC, 1995; ATSDR, 1997; New York State Department of Health, 1997; U.S. EPA, 1985, 1986, 1991; CAL/EPA, 2000), but will focus on more recent developments in the areas of metabolism and liver and kidney toxicity.

	II. Pathways of Perchloroethylene Metabolism

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A. Cytochrome P450-Dependent Oxidation and Associated Enzymes

1. Overview of Cytochrome P450-Dependent Pathway. The overall scheme of Perc metabolism by the P450 pathway is shown in Fig. 1. The initial step is catalyzed by P450 and is believed to yield Perc-epoxide (metabolite 2) as the initial metabolite. There does not appear to be the controversy about the existence of an epoxide intermediate for Perc, unlike that for the related compound TRI (Miller and Guengerich, 1982, 1983; Cai and Guengerich, 1999, 2000). The epoxide (metabolite 2) generated by the action of P450 on Perc (metabolite 1) may have several fates, including conversion to oxalate dichloride (metabolite 7), trichloroacetyl chloride (metabolite 3), trichloroacetyl aminoethanol (metabolite 8), and chloral (metabolite 12). Both chloral and trichloroacetyl chloride may be converted to trichloroacetic acid (TCA; metabolite 4), which is the predominant metabolite recovered in urine of both humans and rodents (Ohtsuki et al., 1983; Dekant et al., 1987; Birner et al., 1996; Völkel et al., 1998). Additional metabolites are derived from these metabolites, and include dichloroacetic acid (DCA; metabolite 10), monochloroacetic acid (metabolite 11), trichloroethanol (TCOH; metabolite 5), and its glucuronide (TCOG; metabolite 6), and oxalate (metabolite 9). After excretion from the liver in the bile, TCOG may undergo significant enterohepatic recirculation (Stenner et al., 1997), being cleaved and thereby regenerating TCOH in the liver. TCOH can also generate TCA. A significant portion of Perc is completely metabolized to CO₂ in a dose-dependent manner (Pegg et al., 1979; Schumann et al., 1980; Frantz and Watanabe, 1983). Because no complete balance study of end metabolites of Perc has been reported in humans, it is possible that all metabolites of Perc have not been identified. Several Perc metabolites are also formed in the oxidative metabolism of TRI (for reviews on TRI metabolism, see Davidson and Beliles, 1991; Goeptar et al., 1995; Lash et al., 2000a). However, the relative importance of individual metabolites varies considerably between the two compounds (Birner et al., 1996). Overall, the data indicate that TCOH, its glucuronide, and their precursor, chloral, are quantitatively less important to Perc metabolism than TCA and its epoxide and trichloroacetyl chloride precursors.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Metabolism of Perc by the P450 pathway. *Identified urinary metabolites: 1, Perc; 2, Perc epoxide; 3, trichloroacetyl chloride; 4, trichloroacetate; 5, trichloroethanol; 6, trichloroethanol glucuronide; 7, oxalate dichloride; 8, trichloroacetyl aminoethanol; 9, oxalate; 10, dichloroacetate; 11, monochloroacetate; 12, chloral.

2. Role of Specific Cytochrome P450 Enzymes in Perchloroethylene Metabolism. There is little direct information on the role of specific enzymes in the oxidative metabolism of Perc. Presumably, CYP2E1 plays a significant role in Perc metabolism in rodent liver and kidney and human liver, because this P450 enzyme has a substrate specificity that includes TRI and a variety of other small, halogenated solvents (Guengerich and Shimada, 1991; Guengerich et al., 1991). Although rat kidney expresses CYP2E1 (Ronis et al., 1998; Cummings et al., 1999), human kidney does not appear to express this enzyme (Amet et al., 1997; Cummings et al., 2000b). The liver is quantitatively the predominant site of oxidative metabolism of Perc, although P450s that can metabolize Perc are present to varying degrees in most tissues. Renal oxidative metabolism of Perc by CYP2E1 is, therefore, relevant only for rodents. Other enzymes (including other P450s) may be involved, however, and these can take the place of CYP2E1 in metabolizing Perc. Costa and Ivanetich (1980) showed that hepatic metabolism of Perc in male Long-Evans rats was increased by pretreatment of rats with pregnenolone-16-carbonitrile or phenobarbital, which induce expression of CYP3A1 and CYP2B1/2, respectively. Pregnenolone-16-carbonitrile increased Perc P450 metabolism by about 70%, whereas phenobarbital produced a 2.6-fold increase in Perc metabolism by P450. Hence, CYP2B1/2 appears to be the major contributor (presumably in addition to CYP2E1) toward oxidative metabolism of Perc. Interestingly, chlorzoxazone and p-nitrophenol were originally considered to be selective CYP2E1 substrates, but recently have been shown to undergo significant metabolism by CYP3A enzymes (Jayyosi et al., 1995; Gorski et al., 1997; Zerilli et al., 1997). Thus, there is precedence for CYP3A enzymes (CYP3A1 or CYP3A2 in the rat and CYP3A4 in humans) metabolizing substrates that are considered to be specific or selective for CYP2E1. The broad range of halogenated hydrocarbons and other small organic molecules that undergo oxidation by CYP2E1 and the existence of several drugs and physiological or pathological conditions that may lead to induction of CYP2E1 suggest that certain conditions or prior or concurrent exposure to other chemicals, such as ethanol or acetaminophen, may alter the metabolism and hence the toxic response to Perc.

4. Species Differences in Cytochrome P450-Dependent Metabolism of Perchloroethylene. Species differences exist in the rates of P450-dependent metabolism of many substrates, including Perc. Völkel et al. (1998) compared the metabolism of Perc to either oxidative or GSH-derived metabolites in rats and human volunteers exposed to Perc by inhalation. Humans were exposed to 10, 20, or 40 ppm of Perc for 6 h in an exposure chamber, and rats were similarly exposed to 10, 20, 40, or 400 ppm of Perc for 6 h. TCA was the major urinary metabolite in both species; however, the rate of excretion of TCA was markedly slower in humans than in rats. The elimination half-time of TCA in the urine was approximately 4.1-fold longer in humans than in rats (45.6 versus 11.0 h). Maximal TCA concentrations in blood were 3- to 10-fold lower (depending on dose) in humans than in rats exposed to 10 or 40 ppm of Perc. Humans exposed to 10 or 40 ppm of Perc for 6 h had TCA concentrations in blood after 24 h of 0.45 and 3.04 nmol/ml of plasma, respectively, whereas rats exposed to 10 or 40 ppm of Perc for 6 h had TCA concentrations in blood after 24 h of 4.24 and 9.86 nmol/ml of plasma, respectively. DCA was not detectable in human urine, but was detected in rat urine at cumulative levels that were approximately 10% of those of TCA (approximately 200 nmol and 2 µmol for DCA and TCA, respectively, after 80 h, from a 6-h exposure to 40 ppm Perc).

B. Glutathione Conjugation Pathway

1. Overview of Glutathione Conjugation Pathway. Besides P450-dependent metabolism, which occurs predominantly in the liver, Perc undergoes conjugation with GSH, which is catalyzed by GSTs, to form S-(1,2,2-trichlorovinyl)glutathione (TCVG). This is the initial step in the pathway that leads to formation of a reactive metabolite that is associated with toxic effects in the kidneys (see Section VII.). Figure 2 illustrates the pathway leading from GSH conjugation of Perc to generation of reactive metabolites by the cysteine conjugate -lyase (-lyase) and other enzymes or to a nontoxic mercapturate excretory product. After formation of TCVG (metabolite 2), which occurs predominantly in the liver, but is also known to occur in the kidneys (Lash et al., 1998a), TCVG is processed by -glutamyltransferase (GGT) and cysteinylglycine dipeptidase to the corresponding cysteine S-conjugate S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) (metabolite 3). The enzymatic activities responsible for the metabolism of TCVG are also present in tissue besides the kidneys, such as the brain, suggesting the possibility that reactive metabolites may be formed in tissues besides the primary target organ.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Metabolism of Perc by the glutathione conjugation pathway. *Identified urinary metabolites: 1, Perc; 2, TCVG; 3, TCVC; 4, NAcTCVC; 5, NAcTCVC sulfoxide; 6, 1,2,2-trichlorovinylthiol; 7, TCVCSO; 8, 2,2-dichlorothioketene; 9, dichloroacetate. Enzymes: GST, GGT, dipeptidase (DP), -lyase, FMO3, CCNAT, CYP3A1/2, and CYP3A4. Unstable, reactive metabolites are shown in brackets.

2. Glutathione S-Transferases. Because the first step of the GSH-dependent pathway of Perc metabolism is catalyzed by GSTs, sex- or species-dependent differences in this step may play a significant role in determining overall flux and thus generation of reactive and toxic metabolites. There is limited information available on differential expression and activity of the various GST isoenzymes. With specific regard to Perc metabolism, however, no direct information is available on the role of specific isoenzymes in TCVG formation, although some data are suggestive of a function for certain isoenzymes. GSTs are a family of isoenzymes (Mannervik, 1985) that are found in the cytoplasmic compartment of cells in most tissues, with the highest amounts of total GST protein found in the liver. Cytoplasmic GSTs are grouped into seven classes (, µ, , , , , and Z, but denoted as A, M, P, T, and Z in humans), based on primary structure, substrate selectivity, sensitivity to inhibitors, and immunological properties. Only GST/A, µ/M, /P, /T, and Z/Z are relevant for mammalian liver and kidney. Additionally, a distinct microsomal GST isoenzyme is found in most tissues (Otieno et al., 1997). A summary of selected properties of the five mammalian GST isoenzymes in the cytoplasm is presented in Table 1.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Perc metabolism to TCVG in the liver and kidney subcellular fractions from male and female F344 rats (A) and B6C3F1 mice (B). Subcellular fractions were obtained by differential centrifugation of homogenates and were incubated for 60 min with 2 mM Perc and 5 mM GSH. TCVG formation was measured after derivatization of acid extracts with iodoacetate and 1-fluoro-2,4-dinitrobenzene, separation by ion-exchange, gradient high-performance liquid chromatography on an amine column using a methanol-acetate mobile phase, and absorbance detection of N-dinitrophenyl-TCVG at 365 nm. The limit of detection is ~50 pmol. Results are means ± S.E. of measurements from three separate tissue preparations.

3. -Glutamyltransferase. TCVG, like other GSH conjugates, is processed by GGT to the cysteinylglycine conjugate S-(1,2,2-trichlorovinyl)-L-cysteinylglycine and then by dipeptidases to form TCVC (Lash et al., 1988). It is these two steps that provide substrate for the actual bioactivation enzymes, which generate the reactive and toxic metabolites. The tissue distribution of GGT activity is a major determinant of the handling of GSH conjugates, including TCVG, and is thus an important factor in the renal specificity of action of nephrotoxic GSH S-conjugates. GGT is the only enzyme that can cleave the -glutamyl bond found in GSH and GSH S-conjugates, and is localized on the luminal, or brush-border, plasma membrane of epithelial cells, such as those in the renal proximal tubule, small intestine, and biliary duct. GGT is also an ectoenzyme, with its active site facing outside the cell. Hence, GSH or GSH S-conjugates must be in the luminal space of the epithelium to be degraded.

4. Cysteine Conjugate -Lyase. As stated above, formation of the cysteine conjugate TCVC represents a branch point in this metabolic pathway, because TCVC can be both bioactivated and detoxified by different enzymes. The enzyme that has received the most attention and is the primary one responsible for renal bioactivation of nephrotoxic cysteine S-conjugates is the -lyase. The -lyase catalyzes either a -elimination reaction, releasing the thiol, pyruvate, and ammonia, or a transamination reaction that produces the corresponding -keto acid, which subsequently rearranges spontaneously to release the thiol and pyruvate (Elfarra et al., 1987). The -lyase is a family of pyridoxal phosphate-containing enzymes that are found in several tissues besides the kidneys, including rat and human liver (Tateishi et al., 1978; Dohn and Anders, 1982; Stevens and Jakoby, 1983; Stevens, 1985a; Tomisawa et al., 1986), intestinal microflora (Tomisawa et al., 1984; Larsen, 1985; Larsen and Stevens, 1986), and rat brain (Alberati-Giani et al., 1995; Malherbe et al., 1995). It is important to note, however, that many of these various -lyase activities are catalyzed by distinct enzymes with varying substrate specificities or are not exposed to cysteine conjugates in their normal processing. Thus, although the liver contains significant -lyase activity as a catalytic function of kynureninase (Stevens, 1985a), this activity plays little role, if any, in metabolism or toxicity because the liver is not exposed to significant amounts of cysteine conjugates in a way that would lead to intrahepatic bioactivation. No liver pathology is observed after treatment of rats with DCVG or DCVC, the GSH and cysteine conjugates, respectively, of TRI (Dohn and Anders, 1982; Elfarra and Anders, 1984; Elfarra et al., 1986). This presumably would be the case for TCVG or TCVC, although this has not been specifically tested.

View this table: [in this window] [in a new window]   TABLE 2 Sex and species-dependent differences in kinetics of renal cytoplasmic cysteine conjugate -lyase with TCVC as substrate

5. Other Reactions of S-(1,2,2-trichlorovinyl)-L-cysteine and Evaluation of Relative Rates of Each Step of the Glutathione Conjugation Pathway. As described in Section II.B.1., TCVC can be viewed as representing a branch point in the overall pathway of GSH S-conjugate metabolism (cf. Fig. 2). Besides metabolism by the -lyase, TCVC may be bioactivated by either FMO3 or P450 to form TCVCSO, or is N-acetylated to form the mercapturate, which can be readily excreted. TCVCSO may also be a substrate for the -lyase, although, as noted above, it is likely to be a very poor substrate so that most of the bioactivation occurs by spontaneous decomposition. NAcTCVC can also be deacetylated by an acylase to regenerate TCVC, or can be oxidized to NAcTCVC sulfoxide by CYP3A enzymes. Both TCVCSO and N-acetyl-S-(1,2,2-trichlorovinyl-L-cysteine sulfoxide may generate highly reactive and cytotoxic alkylating species. Viewing the cysteine conjugate as a branch point brings one readily to the conclusion that the extent of toxicity will be determined largely by two major factors: 1) the chemical reactivity of the product of the -lyase reaction or the sulfoxide; and 2) the balance between flux through the -lyase-FMO3 pathways and CCNAT/acylase-CYP3A pathways. It is this balance that determines how much reactive metabolite is formed.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Analysis of risk or susceptibility to renal injury based on fluxes of cysteine conjugate formation and metabolism. Risk for nephrotoxicity or other toxic effects with the kidneys as a target organ can be calculated from apparent rate constants (k) for each enzymatic step involved in the formation and further metabolism of the cysteine conjugate. In this case, (GSH)-derived metabolites of Perc are used, but this approach should apply to other halogenated alkanes or alkenes that form potentially nephrotoxic metabolites by these enzymes. TCVSH, 1,2,2-trichlorovinylthiol; NAcTCVCSO, NAcTCVC sulfoxide. Forward rate constants (k) are defined for GST, -lyase, CCNAT, FMO3, acylase, and CYP3A.

k-lyase

6

4

Perc appears to be a much poorer substrate than its congener TRI for P450 (Ohtsuki et al., 1983; Völkel et al., 1998). In vivo, Perc is conjugated with GSH more extensively (1 to 2% of the dose) (Dekant et al., 1986a) than TRI (<0.005% of the dose) (Green et al., 1997). These differences are replicated in in vitro studies, where conjugation of Perc with GSH occurs at faster rates than TRI in mice and rats (Lash et al., 1995, 1998a,b). In experiments to assess the effects of modulation of P450 expression and/or activity on GSH conjugation of Perc, induction of hepatic or renal CYP2E1 by pretreatment of male F344 rats with pyridine significantly diminished rates of TCVG formation (L. H. Lash, W. Qian, P. Huang, A. A. Elfarra, and J. C. Parker, unpublished data). These results suggest that P450 effectively competes with GST for metabolism of Perc. Indeed, Dekant et al. (1987) showed that, in incubations of rat liver microsomes with either Perc or 1-chloro-2,4-dinitrobenzene as substrate, formation of the GSH conjugate was reduced by 70 to 85% by inclusion of NADPH. Although the same type of experiment has not been performed with human tissue, Lash et al. (1999) examined the interaction between GST and P450 in human liver microsomes in the metabolism of TRI: inclusion of NADPH in incubations of human liver microsomes with GSH and TRI significantly reduced formation of DCVG, compared with incubations without NADPH. In contrast, inclusion of GSH in incubations of human liver microsomes with TRI and NADPH had no significant effect on chloral hydrate formation, compared with that in the absence of GSH. Hence, P450 can successfully compete with GST for metabolism of TRI, but GST is ineffective in competing with P450, due to lower affinity and specific activity.

One may conclude that a low-affinity, low-activity pathway (i.e., GSH conjugation) is only of toxicological significance at high doses and/or when the high-affinity pathway (i.e., P450) is saturated. Indeed, this conclusion has been made for both Perc and TRI and particularly for humans, compared with rodents (Green, 1990; Green et al., 1990, 1997; Völkel et al., 1998). This is based in part on kinetics, but also on the relatively low recovery of urinary mercapturates, compared with urinary TCA and related P450-derived metabolites (Green et al., 1990; Birner et al., 1996). Thus, urinary mercapturates comprise from approximately 1% to as little as 0.03% of total recovered urinary metabolites. As far as the kinetic argument is concerned, it is critical to understand that the GSH conjugation pathway yields highly reactive and chemically unstable metabolites, whereas the products of the P450 pathway that are associated with toxicity (e.g., TCA and DCA) are by and large chemically stable, although their relatively unstable precursors (i.e., Perc-epoxide and trichloroacetyl chloride) may also be involved. This is even truer for Perc as substrate than it is for TRI (Lash et al., 1998a). Thus, relatively small amounts of a reactive metabolite may be required to elicit significant biochemical effects in the target cell, making arguments based simply on amounts of metabolites formed or isolated without logical foundation.

As far as the argument that mercapturates can be used as a measure of flux through the GSH conjugation pathway, this is also without firm foundation, as discussed above. For the mercapturate to be a valid indicator of the generation of toxic metabolites, one would have to know precisely, over a wide range of relevant substrate concentrations, the quantitative relationship between the various enzymatic steps that metabolize the cysteine and N-acetylcysteine conjugates (see Fig. 4). This is exceedingly difficult because many of the metabolites are reactive and are not readily recoverable, either because they form covalent adducts with cellular macromolecules or because they are chemically reactive and not easily quantified. Hence, urinary mercapturates can be validly used as an indicator of exposure, providing evidence of pathway operation to a point, and substrate availability for further processing. Based on the amounts of excretion, however, mercapturates clearly would not be a very sensitive indicator of the amount of toxic products formed. Moreover, no inferences concerning the generation of nephrotoxic and potentially nephrocarcinogenic species should be made from measurements of urinary mercapturates. It is clear, however, that the GST pathway is generally more quantitatively significant in rats than in humans, but that irrespective of species, the relative role of this pathway in Perc metabolism is clearly greater than it is for TRI.

	III. Physiologically Based Pharmacokinetic Models for Perchloroethylene

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PBPK models have been useful for interspecies extrapolations when, for example, data from humans are lacking. These models can also be useful in determining the influence of changes in specific parameters or physiological functions on the disposition of given chemicals of interest. For example, a PBPK model for Perc developed by Clewell and colleagues (Gearhart et al., 1993