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IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Some Chemicals Used as Solvents and in Polymer Manufacture. Lyon (FR): International Agency for Research on Cancer; 2017. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 110.)

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Some Chemicals Used as Solvents and in Polymer Manufacture.

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4Mechanistic and Other Relevant Data

4.1. Toxicokinetic data

4.1.1. Absorption

(a) Humans

Dichloromethane is a lipophilic solvent of low relative molecular mass, which can readily cross biological membranes. Pulmonary uptake is rapid, approaching steady state within a few hours after the start of exposure (Riley et al., 1966; DiVincenzo et al., 1971, 1972; Astrand et al., 1975; DiVincenzo & Kaplan, 1981). Measured values of pulmonary uptake are about 55–75% at rest and 30–40% during physical exercise (Astrand et al., 1975; DiVincenzo & Kaplan, 1981). The blood:air partition coefficient for dichloromethane describes the ratio of the concentrations in the two media at steady state, and is a factor in determining pulmonary uptake. The partition coefficient has been measured in vitro using vial equilibrium methods. Mean reported values range from around 8 to 10 for humans (Sato & Nakajima, 1979; Gargas et al., 1989; Meulenberg & Vijverberg, 2000). However, these data might have been influenced by the presence of glutathione S-transferase T1 (GSTT1) in human erythrocytes (Schröder et al., 1996).

Data on oral absorption in humans are limited to case reports of accidental ingestion, and suggest that dichloromethane is also readily absorbed by this route of exposure (Hughes & Tracey, 1993; Vetro et al., 2012). Quantitative estimates of oral bioavailability in humans are not available because the ingested amounts are not known precisely.

Ursin et al. (1995) report that the permeability of human skin to dichloromethane is 24 g/m2 per hour. No other information on human dermal absorption of dichloromethane was available to the Working Group.

(b) Experimental systems

Inhalation studies in experimental animals provide clear evidence that dichloromethane is readily absorbed via the lungs into the systemic circulation (Carlsson & Hultengren, 1975; Anders & Sunram, 1982; McKenna et al., 1982; Andersen et al., 1991). The blood:air partition coefficient for dichloromethane, measured in vitro using vial equilibrium methods, has been reported to range from 19 to 23 for rodents (Gargas et al., 1989; Marino et al., 2006).

Absorption from the gut after oral doses is rapid and nearly complete, according to reports of several studies with radiolabel in mice and rats (McKenna & Zempel, 1981; Angelo et al., 1986a, b). For instance, Angelo et al. (1986b) reported that on average 97% of the radiolabel was recovered in expired air as dichloromethane, CO, and carbon dioxide (CO2) in the 24 hours after each repeated oral dose of 50 or 200 mg/kg per day in rats. Angelo et al. (1986a) reported absorption in mice to be more rapid (but equally extensive) with an aqueous vehicle than with an oil-based vehicle, consistent with studies on other chlorinated solvents.

No studies of dermal uptake of dichloromethane in experimental animals were available to the Working Group.

4.1.2. Distribution and body burden

(a) Humans

Once absorbed, dichloromethane enters blood circulation and undergoes rapid systemic distribution to tissues. The highest concentrations are expected in adipose tissue and other fatty tissues, due to the lipophilicity of the compound. Engström & Bjurström (1977) detected dichloromethane in fat biopsy specimens obtained from men exposed to dichloromethane for 1 hour during light exercise. Other data in humans on tissue distribution in vivo are limited to tissues taken from autopsies after accidental fatalities, which showed wide systemic distribution in blood and across all tested tissues, including the fat, lung, liver, heart, kidney, spleen, and brain (Moskowitz & Shapiro, 1952; Winek et al., 1981; Shinomiya & Shinomiya, 1985; Manno et al., 1989; Leikin et al., 1990; Kim et al., 1996; Goullé et al., 1999). Goullé et al. (1999) and Leikin et al. (1990) measured the largest number of tissues, and found the highest concentrations in brain, spleen, and fat.

Engström & Bjurström (1977) also measured an in-vitro partition coefficient of 51 between adipose tissue and air using a vial equilibrium method. This value is about five times the blood:air partition coefficient, consistent with the lipophilicity of dichloromethane. Partition-coefficient measurements for other human tissues were not available to the Working Group.

(b) Experimental systems

Studies in experimental animals provide clear evidence that dichloromethane distributes widely to all tissues of the body. After in-vivo oral and/or intravenous exposures in mice and/or rats, dichloromethane has been measured in the liver, kidney, lung, and whole carcass, with the highest concentrations in the liver (Angelo et al., 1986a, b). Several inhalation experiments with radiolabeled dichloromethane detected the presence of radiolabel in all tissues, including the liver, kidney, adrenals, brain, fat, lung, muscle, and testes (Carlsson & Hultengren, 1975; McKenna et al., 1982). While part of the radiolabel is likely to be metabolites, it is likely that a substantial portion also represents dichloromethane. Experiments in animals show that dichloromethane readily crosses the blood–brain barrier and the placenta (Savolainen et al., 1981; Anders & Sunram, 1982).

Tissue:air partition coefficients have also been measured in vitro for several tissues in rats and mice, including fat, liver, muscle, skin, kidney, and brain (Andersen et al., 1987; Gargas et al., 1989; Clewell et al., 1993). The highest reported values are for fat (60–120), with values for the remaining tissues ranging from 8 to 40, as compared with blood:air partition coefficients of around 20.

4.1.3. Metabolism

(a) Overview

The pathways for metabolism of dichloromethane were initially characterized nearly 40 years ago in the mid-1970s and are widely considered to be well established (Kubic & Anders, 1975, 1978; Ahmed & Anders, 1976, 1978). Dichloromethane is metabolized by either of two pathways, as summarized in Fig. 4.1.

Fig. 4.1

Fig. 4.1

Pathways for the metabolism of dichloromethane

One pathway, a reductive dehalogenation that is a mixed-function oxygenation, was subsequently shown to be catalysed by cytochrome P450 2E1 (CYP2E1) (Guengerich et al., 1991), and ultimately generates CO and CO2 as stable end products. The initial product of the reaction, chloromethanol, spontaneously rearranges to form formyl chloride, which is reactive and can spontaneously generate CO or react with nucleophiles such as glutathione (GSH) to generate formylglutathione; the latter rearranges to release CO2. CO avidly reacts with haemoglobin, displacing oxygen and forming COHb.

The other pathway for dichloromethane metabolism involves conjugation with GSH, forming S-chloromethyl GSH. The conjugation is catalysed by GSTs, with the GSTT1 isoform being the most active (Mainwaring et al., 1996; Sherratt et al., 1997). S-Chloromethyl GSH is reactive and is believed to be one of the dichloromethane metabolites responsible for DNA binding and mutagenicity (Graves & Green, 1996). Alternatively, S-chloromethyl GSH can be hydrolysed to form hydroxymethyl GSH, which can either decompose to release formaldehyde or be oxidized by formaldehyde dehydrogenase to form S-formyl GSH. The latter is subsequently hydrolysed to release formic acid and GSH. Formic acid further decomposes to release CO2. Thus, while both the CYP and GST pathways can generate CO2, only the CYP pathway produces CO from dichloromethane. Although both pathways can generate reactive and unstable metabolites that are mechanistically linked to dichloromethane-induced genotoxicity and carcinogenesis, it is thought that these come primarily from the GST pathway (Andersen et al., 1987).

Despite the wealth of data over more than three decades from in-vivo and in-vitro studies in humans and experimental animals, which supports the function of both CYP2E1 and GSTT in dichloromethane metabolism, Evans & Caldwell (2010a) proposed a different explanation for dichloromethane metabolism that involves only CYP2E1. As precedent for this alternative metabolic pathway, the authors cited studies by Harrelson and colleagues (Harrelson et al., 2007, 2008) and Tracy (2006) and two studies by Guengerich and colleagues (Watanabe & Guengerich, 2006; Watanabe et al., 2007). Their conclusion was that the available data support a limited role for GST-dependent metabolism. Anders et al. (2010) criticized this proposal by noting that the authors misinterpreted the data from Watanabe & Guengerich (2006) and Watanabe et al. (2007), for the limited in-vitro data supporting the alternative mechanism, for dismissing the wealth of data on the role of GSTT in dichloromethane metabolism, mutagenicity, and carcinogenicity, and for rejecting without any sound basis the several well-established and validated physiologically based pharmacokinetic models of dichloromethane metabolism in humans and rodents. Although Evans & Caldwell (2010b) maintained the validity of their interpretations, no data supporting metabolism of dichloromethane that exclude GST, particularly at higher dichloromethane concentrations, were identified by the Working Group.

Specific studies on dichloromethane metabolism and mechanisms in humans and human-derived tissues and in experimental systems are summarized below.

(b) Humans or human-derived tissues

Oxidative metabolism of dichloromethane to CO was first demonstrated in occupationally exposed humans (Stewart et al., 1972a, b; Ratney et al., 1974; Astrand et al., 1975). DiVincenzo & Kaplan (1981) measured dichloromethane metabolism using COHb in nonsmoking volunteers exposed to dichloromethane vapour at concentrations of up to 200 ppm for 7.5 hours (once, or daily for 5 days). Dose-dependent COHb formation was readily demonstrated, with the single-day exposures resulting in peak CoHb saturations of 1.9%, 3.4%, 5.3%, and 6.8%, respectively, at 0, 50, 100, and 200 ppm. A comparative study of the effects in humans exposed to either CO or dichloromethane up to concentrations that produce 5% COHb saturation was performed; both substances impaired performance (Putz et al., 1979), this was consistent with evidence that about 70% of dichloromethane at relatively low doses is metabolized to CO (Andersen et al., 1991).

Metabolic parameter estimates made by Clewell (1995) show that the oxidative pathway in human liver has a capacity of 100- to 200-fold that of the GST pathway, although in-vitro studies by Reitz et al. (1989) generally showed a much more modest difference in capacity of the two pathways, with the CYP pathway having a two- to fourfold higher capacity than the GST pathway in most of the human liver samples studied.

Bogaards et al. (1993) measured GST activity with dichloromethane and 1-chloro-2,4-dinitrobenzene (CDNB) in nine human liver cytosol samples, finding three distinct activity groups. Specifically, with dichloromethane, two exhibited no detectable activity, four exhibited relatively low activity (0.2–0.4 nmol/min per mg protein), and three exhibited relatively high activity (0.9–1.1 nmol/min per mg protein). Interestingly, although metabolic activity with CDNB as substrate also exhibited an approximately fivefold variation among the nine samples, there were no apparent null variants and the pattern of metabolism with CDNB and dichloromethane did not coincide. While CDNB is a substrate for multiple GST isoforms (Habig et al., 1974), it is now widely accepted that dichloromethane is selectively metabolized by GSTT (see below).

Mainwaring et al. (1996) determined mRNA and protein expression of GSTT1 in cells from human liver and lung, both of which are target organs for dichloromethane in the mouse. While expression of GSTT1 was readily detected in the liver, very low levels were detected in the lungs. Furthermore, GSTT1 activity with dichloromethane was measured in three samples of lung at 0.06, 0.21, and 0.23 nmol/min per mg protein, which was about one order of magnitude less than that in human liver.

Casanova et al. (1997) detected RNA–formaldehyde adducts in human hepatocytes with functional GST genes and incubated with dichloromethane, which is evidence that formaldehyde is formed in human cells as a metabolite of dichloromethane.

GST activity in human liver was further related to carcinogenic risk with dichloromethane in studies of GSTT1 polymorphism (El-Masri et al., 1999; Sherratt et al., 2002; Olvera-Bello et al., 2010). Although the importance of genetic polymorphisms in determining carcinogenic risk is discussed elsewhere (see Section 4.5.1), it is mentioned here as providing further evidence of the presence and importance of GST activity in dichloromethane metabolism.

In addition to absolute levels of GSTT protein expression in target organs, another important issue is the subcellular localization of the expressed enzyme. While GSTT11 in mouse liver is readily found in cytoplasm and nuclei of hepatocytes, it is found at lower levels in nuclei of bile-duct epithelial cells, and in cytoplasm and nuclei of some human hepatocytes (Sherratt et al., 2002). This less intense nuclear localization is thought to be of significance for carcinogenic risk because less S-chloromethyl GSH and formaldehyde will be generated near DNA.

GST is also present in human erythrocytes and is thought to play a role in toxicity of dichloromethane in lymphocytes (Hallier et al., 1993, 1994). Erythrocyte GSTT is polymorphic, as further discussed in Section 4.5.

(c) Experimental systems

(i) Rat

The metabolism of dichloromethane has been extensively studied in several experimental systems, predominantly those derived from rodents. This is particularly important in that mouse liver and lung have been identified as prominent target organs for dichloromethane, and toxicity has been clearly linked to metabolism. Some of the earliest studies that established the basic outlines of dichloromethane metabolism were conducted in rat liver microsomes (Kubic & Anders, 1975, 1978), rat liver cytosol (Ahmed & Anders, 1976, 1978), and rat lung microsomes (Kubic & Anders, 1975).

As noted above, the CYP-dependent oxidative pathway is considered to be a high-affinity, low-capacity pathway for dichloromethane metabolism, while the GST pathway is a low-affinity, high-capacity pathway. An in-vivo study of metabolism after oral administration of 14C-labelled dichloromethane in rats showed dose-dependent metabolism primarily to CO and CO2, with clear evidence of saturation (McKenna & Zempel, 1981). While rats given a dose of dichloromethane at 1 mg/kg metabolized approximately 88% of the administered dose over 48 hours, those given dichloromethane at 50 mg/kg only metabolized about 28% of the administered dose over the same period. Saturation of dichloromethane metabolism after inhalation in rats was also demonstrated by Kurppa & Vainio (1981), who showed that blood COHb levels stabilized at dichloromethane exposures of 500 ppm.

Gargas et al. (1986) measured COHb levels in rats given dichloromethane or other dihalomethanes by inhalation in a closed-atmosphere exposure system. The bromine-containing dihalomethanes exhibited the highest activities, while fluorine-containing dihalomethanes exhibited no detectable activity. Maximal rates of COHb formation from dibromomethane, chlorobromomethane, and dichloromethane were 72, 54, and 47 µmol/kg per hour, respectively. Pretreatment with pyrazole, which inhibits microsomal oxidation, abolished production of CO. Depletion of GSH with 2,3-epoxypropanol increased the steady-state levels of COHb generated from dichloromethane.

Takano & Miyazaki (1988) applied dichloromethane to perfused livers of male Wistar rats previously given phenobarbital to induce CYP, and examined spectral changes by scanning reflectance spectrophotometry. Both with and without addition of exogenous CO, a type-I spectral change with a peak at 450 nm was observed, demonstrating CYP-dependent metabolism of dichloromethane to CO in the intact rat liver.

Kim & Kim (1996) further explored the role of CYP2E1 in dichloromethane metabolism by examining the effect of prior administration of organic solvents that induce CYP2E1 on COHb levels in adult female rats after intraperitoneal administration of dichloromethane (3.0 mmol/kg). Peak COHb levels in blood reached 21%, 16%, and 23% in rats pretreated with benzene, toluene, or m-xylene, respectively, compared with only about 10% in rats given dichloromethane alone. The selective CYP2E1 inhibitor disulfiram (3.4 mmol/kg) blocked the elevations in COHb. No effects on hepatic GSH levels were observed with the single administration of the solvents, indicating no involvement with changes in the GST pathway in the observed responses.

(ii) Mouse

Reitz and colleagues analysed dichloromethane metabolism by the CYP and GST pathways in the liver and lung of male B6C3F1 mice, F344 rats, Syrian golden hamsters, and humans (Table 4.1) (Reitz et al., 1988). Several striking species-dependent differences are clearly evident from the data. First, mice exhibit similar rates of CYP-dependent metabolism in liver as hamsters and nearly threefold higher rates than rats or humans. Second, in lung tissue CYP-dependent metabolism of dichloromethane in mice was ~30-fold higher than in rats and ~5-fold higher than in hamsters. No CYP-dependent metabolism was detected in the human lung sample. Third, even greater species-dependent differences in addition to interindividual differences were observed in the liver and lung for GST-dependent dichloromethane metabolism. In this case, rates of GSH conjugation in mouse liver were ~4-fold faster than in rats, ~20-fold faster than in hamsters, and ~10-fold faster than in humans. Finally, perhaps the greatest species-dependent metabolic difference was observed for GST metabolism in the lung. Here, rates in mice were ~7-fold faster than in rats and ~20-fold faster than in humans. These metabolic differences have been interpreted to explain species-dependent differences as well as interindividual differences in target-organ specificity and sensitivity to dichloromethane-induced mutagenesis and carcinogenicity (Green, 1990; Starr et al., 2006). Furthermore, data on tumour incidence across species show a correlation with the amount of dichloromethane metabolized by GST but not by CYP (Andersen et al., 1987).

Table 4.1. Reaction rates for dichloromethane metabolism by CYP and GST in liver and lung tissue from different species.

Table 4.1

Reaction rates for dichloromethane metabolism by CYP and GST in liver and lung tissue from different species.

Ottenwälder et al. (1989) gave two specific CYP inhibitors (i.e. pyrazole, 320 mg/kg, and diethyldithiocarbamate, 300 mg/kg) to male B6C3F1 mice also exposed to dichloromethane at 1000 or 3000 ppm, or a mixture of dichloromethane at 1000 ppm and methyl chloride at 1000 ppm. For those mice given only dichloromethane, uptake by inhalation was markedly decreased by the CYP inhibitors. In contrast, CYP inhibitors had no effect on the uptake of methyl chloride by inhalation. Because methyl chloride is metabolized solely by GSTs, these results showed that even at relatively high exposures, dichloromethane is predominantly metabolized by CYP. These results contrasted with those of Andersen et al. (1987) described above, who concluded that GST-dependent, rather than CYP-dependent, metabolism was critical for dichloromethane-induced liver tumorigenesis.

The in-vivo metabolism of dichloromethane by CYP was further demonstrated by Casanova et al. (1992), who pre-exposed male B6C3F1 mice to dichloromethane at 4000 ppm for 6 hours per day for 2 days, and then on day 3 to 14C-labelled dichloromethane at a declining concentration (4500–2500 ppm). DNA–protein cross-links and incorporation of 14C derived from dichloromethane into DNA was observed in the liver of these mice.

Foster et al. (1994) also showed that modulation of pulmonary CYP activity can also alter responses of the lung to dichloromethane.

4.1.4. Excretion

(a) Humans

In humans, the main route of excretion of dichloromethane is by exhalation of the parent compound and its primary metabolites CO2 and CO, with lesser amounts as dichloromethane excreted in the urine (DiVincenzo et al., 1971, 1972; DiVincenzo & Kaplan, 1981). DiVincenzo & Kaplan (1981) estimated that only 5% of absorbed dichloromethane is exhaled unchanged, 25–34% excreted converted as CO, and the balance excreted as CO2. After cessation of exposure, the half-life of dichloromethane in the blood has been estimated to be about 40 minutes, with concentrations of parent and metabolites returning the preexposure levels within a few days (DiVincenzo et al., 1972; DiVincenzo & Kaplan, 1981). Urinary excretion occurs mostly during and/or within the first hour after cessation of exposure, and in total accounts for less than 0.1% of uptake (DiVincenzo et al., 1971, 1972).

(b) Experimental systems

As in humans, the main route of excretion of dichloromethane in experimental animals is by exhalation of the parent compound and its primary metabolites CO2 and CO, with lesser amounts excreted in the urine. As exposure levels increase, the percentage excreted as unchanged parent compound increases, reflecting saturation of metabolism. For instance, McKenna et al. (1982) reported that in rats exposed to dichloromethane at 50 ppm via inhalation, elimination in expired air consists of about 5% parent compound, and 26% and 27% CO2 and CO, respectively. At 500 and 1500 ppm, elimination of parent compound increased to 30% and 55%, respectively, with declines in the amount of CO2 and CO expired. Similarly, for oral doses of 1 mg/kg, McKenna & Zempel (1981) reported that rats exhaled 12% of the administered dose as parent compound, and 35% and 31% as CO2 and CO, respectively. At higher oral doses (50 mg/kg or greater), rats and mice exhale greater amounts as parent compound (60–80%), and lesser amounts as CO2 and CO (McKenna & Zempel, 1981; Angelo et al., 1986a, b).

Overall, experimental studies in rodents have found that > 90% of absorbed dichloromethane is eliminated within 24 or 48 hours of exposure, regardless of dose. McKenna et al. (1982) reported that after inhalation exposure in rats, a low percentage of the initial body burden of dichloromethane remained at 48 hours. After a single intravenous dose in mice, Angelo et al. (1986a) reported 92–94% recovery within 4 hours after dosing. After repeated oral exposures in mice, Angelo et al. (1986a) reported 90–96% recovery of within 24 hours after each dose.

4.2. Genetic and related effects

Dichloromethane has been studied for genotoxic potential in a variety of assays. The genotoxicity of dichloromethane has been reviewed previously by the Working Group (IARC, 1999).

4.2.1. Humans

(a) In vivo

No data were available to the Working Group.

(b) In vitro

See Table 4.2

Table 4.2. Studies of genotoxicity with dichloromethane in human cell lines in vitro.

Table 4.2

Studies of genotoxicity with dichloromethane in human cell lines in vitro.

Dichloromethane did not induce DNA single-strand breaks in human primary hepatocytes (Graves et al., 1995). There was no induction of DNA–protein cross-links in vitro in human hepatocytes with functional GSTT1 genes (Casanova et al., 1997) or unscheduled DNA synthesis in AH fibroblasts (Jongen et al., 1981) after treatment with dichloromethane in vitro.

In a study by Doherty et al. (1996), dichloromethane induced the formation of kinetochore-staining micronuclei (which are indicative of aneuploidy) and kinetochore-negative micronuclei in human MCL-5 cells that stably express cDNA encoding human CYP1A2, CYP2A6, CYP3A4, CYP2E1, and epoxide hydrolase and in h2E1 cells, which contains a cDNA for CYP2E1. The increased frequency of micronucleus formation is combined with the fact that MCL-5 and h2El cell lines showed the capacity to produce metabolites in the presence of dichloromethane. AHH-1 cells, constitutively expressing CYP1A1, showed no increase in the total frequency of micronucleus formation or in the frequency of kinetochore-staining micronuclei.

Hallier et al. (1993) showed that sister-chromatid exchanges were induced in human peripheral blood lymphocyte cultures from non-conjugator donors lacking GST activity, but not in those from conjugators. This study did not provide details on the type of GST activity that was monitored. Sister-chromatid exchanges were also induced by dichloromethane in vitro in human peripheral blood mononuclear cells (Olvera-Bello et al., 2010). This study also demonstrated that the group with high GSTT1 activity showed a larger increase in the frequency of sister-chromatid exchanges induced by dichloromethane than did the groups with low and medium GSTT1 activity.

4.2.2. Experimental systems

(a) Mammalian systems

See Tables 4.3 and 4.4

Table 4.3. Studies of genotoxicity with dichloromethane in mammalian systems in vivo.

Table 4.3

Studies of genotoxicity with dichloromethane in mammalian systems in vivo.

Table 4.4. Studies of genotoxicity with dichloromethane in mammalian systems in vitro.

Table 4.4

Studies of genotoxicity with dichloromethane in mammalian systems in vitro.

(i) DNA damage

Exposure of B6C3F1 mice to dichloromethane by inhalation induced DNA single-strand breaks in the lung and liver (Graves et al., 1995). Prior treatment of the mice with buthionine sulfoximine (a depletor of GSH) immediately before exposure to dichloromethane reduced the amount of DNA damage to control levels.

Dichloromethane induced DNA single-strand breaks in vivo in AP rat primary hepatocytes and B6C3F1 mouse hepatocytes (Graves et al., 1994b), and in Clara cells (Graves et al., 1995). DNA damage was reduced in Clara cells co-treated with buthionine sulfoximine. DNA single-strand breaks were not observed in the liver or lung of AP rats treated by inhalation (Graves et al., 1994b, 1995), but were induced in the liver of CD rats treated by gavage (Kitchin & Brown, 1994), and in the liver of B6C3F1 mice treated by inhalation (Graves et al., 1994b). Dichloromethane did not cause DNA damage as measured by the comet assay in male B6C3F1 mice exposed by inhalation for 6 weeks (6 hours per day, 5 days per week) at 400, 800, or 1600 ppm (Suzuki et al., 2014).

The frequency of DNA single-strand breaks was increased in vitro in Chinese hamster ovary cells cultured with dichloromethane in the presence, but not in the absence, of an exogenous metabolic activation system (Graves et al., 1994b). DNA single-strand breaks were also induced in Chinese hamster ovary cells exposed to dichloromethane with or without exogenous metabolic activation, the effect being stronger with metabolic activation (Graves & Green, 1996). Conversely, DNA single-strand breaks were not induced in Syrian hamster hepatocytes (Graves et al., 1995).

Hu et al. (2006) performed the standard and proteinase K-modified comet assay to measure DNA damage and DNA–protein crosslinks in untreated V79 cells and in V79 cells transfected with the murine GSTT1 gene (V79 mGSTT1). Dichloromethane induced DNA damage in both cell types. However, the study showed the presence of dichloromethane-induced DNA–protein crosslinks in the V79 mGSTT1 cell line and not in standard V79 cell line, which indicates that GSTT1 was instrumental for the induction of DNA–protein crosslinks. Moreover, dichloromethane formed significantly higher amounts of cytosolic formaldehyde in V79 in GSTT1 cells.

No DNA binding was observed in vivo in the liver or kidney of male rats or male and female mice after intraperitoneal administration of dichloromethane (Watanabe et al., 2007). Covalent binding of dichloromethane to DNA was not observed in the liver, kidney, or lung of rats or mice exposed by inhalation, although metabolic incorporation of 14C was found in normal deoxyribonucleosides in both species (Ottenwälder & Peter, 1989).

DNA–protein cross-links were induced in vivo in the liver, but not the lung of B6C3F1/CrlBR mice exposed to dichloromethane (Casanova et al., 1992). No DNA–protein cross-links were detected in Syrian hamster liver or lung after inhalation of dichloromethane (Casanova et al., 1992). DNA–protein cross-links were not induced in the liver of Syrian golden hamsters, but were observed in the liver of B6C3F1/CrlBR mice treated with dichloromethane by inhalation (Casanova et al., 1996).

Dichloromethane induced DNA–protein cross-links in vitro in hepatocytes of male B6C3F1 mice, but not in hepatocytes of Fischer 344 rats or Syrian hamsters (Casanova et al., 1997). DNA–protein cross-links were also induced in Chinese hamster ovary cells exposed to dichloromethane with or without exogenous metabolic activation, with DNA damage being greater in the presence of metabolic activation (Graves & Green, 1996). Using the proteinase K-modified comet assay, it was demonstrated that dichloromethane induced DNA–protein cross-links in V79 cells transfected with the murine GSTT1 gene, but not in standard V79 cells (Hu et al., 2006). [The Working Group noted that this suggests a key role for GST in genotoxicity induced by dichloromethane.]

In a study in vivo, mice treated with dichloromethane at 2000 ppm [6940 mg/m3] for 6 hours per day, 5 days per week, for 12 weeks showed an increased frequency of sister-chromatid exchange in lung cells (Allen et al., 1990). Exposure to higher concentrations (8000 ppm [27 800 mg/m3] for 2 weeks) also induced an increase in the frequency of sister-chromatid exchange in peripheral blood erythrocytes. Dichloromethane did not induce sister-chromatid exchange in bone marrow of mice treated by intraperitoneal or subcutaneous injection (Westbrook-Collins et al., 1990; Allen et al., 1990). Dichloromethane did not increase the frequency of sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of an exogenous metabolic system (Thilagar & Kumaroo, 1983; Anderson et al., 1990). When tested in Chinese hamster lung V79 cells in the absence of exogenous metabolic activation, dichloromethane induced a slight increase in the frequency of sister-chromatid exchange (Jongen et al., 1981).

Dichloromethane did not induce unscheduled DNA synthesis in vivo in Fischer 344 rats treated by gavage or inhalation, or in B6C3F1 mouse hepatocytes treated by inhalation (Trueman & Ashby, 1987).

(ii) Chromosomal aberration

Dichloromethane did not cause chromosomal aberration in vivo in bone marrow of mice treated by intraperitoneal or subcutaneous injection (Westbrook-Collins et al., 1990; Allen et al., 1990). A small increase in the frequency of chromosomal aberration in mouse bone marrow and lung cells was reported after exposure to dichloromethane at 8000 ppm by inhalation for 6 hours per day, 5 days per week, for 2 weeks (Allen et al., 1990). In a study by Burek et al. (1984), dichloromethane gave negative results in an assay for chromosomal aberration in rat bone marrow.

Dichloromethane induced chromosomal aberration in vitro in Chinese hamster ovary cells in the presence and absence of an exogenous metabolic system in one of two studies (Thilagar & Kumaroo, 1983; Anderson et al., 1990).

(iii) Micronucleus formation

Dichloromethane did not induce micronucleus formation in vivo in bone marrow of mice treated by gavage or intraperitoneal injection (Gocke et al., 1981; Sheldon et al., 1987; Morita et al., 1997). Mice treated with dichloromethane at 2000 ppm [6940 mg/m3] for 6 hours per day, 5 days per week, for 12 weeks showed an increased frequency of micronuclei in peripheral blood erythrocytes (Allen et al., 1990). The highest dose tested (8000 ppm, 6 hours per day, 5 days per week, for 2 weeks) gave positive results in erythrocytes and lung cells, but negative results in bone marrow. On the other hand, dichloromethane did not cause micronucleus formation in male B6C3F1 mice exposed at 400, 800 and 1600 ppm by inhalation for 6 weeks (6 hours per day, 5 days per week) (Suzuki et al., 2014).

(iv) Mutagenicity

Dichloromethane did not cause gene mutation in two inhalation experiments in vivo: a Pig-a assay in male B6C3F1 mice exposed to dichloromethane at 400, 800, or 1600 ppm for 6 weeks (6 hours per day, 5 days per week); and a transgenic rodent gene mutation assay on Gpt Delta C57BL/6J mouse liver treated for 4 weeks (6 hours per day, 5 days per week) with dichloromethane at 800 ppm (Suzuki et al., 2014).

In vitro, dichloromethane was mutagenic in Chinese hamster ovary cells at the Hprt locus in one study, in the presence of exogenous metabolic activation (Graves & Green, 1996), and gave equivocal results in the mouse lymphoma Tk+/– assay in another study (Myhr et al., 1990). DNA sequence analysis of the Hprt mutants of Chinese hamster ovary cells treated with dichloromethane indicated that most mutations were GC→AT transitions (4 out of 8), with two GC→CG transversions and two AT→TA transversions. This pattern was more similar to that of 1,2-dibromoethane (ethylene dibromide) (IARC, 1999) (7 out of 9 being GC→AT transitions) than that of formaldehyde, a metabolite of dichloromethane that has been identified in vitro (see Section 4.1), for which all mutations were single-base transversions and 5 out of 6 arose from AT base pairs (Graves et al., 1996). When tested in Chinese hamster lung fibroblast V79 cells in the absence of exogenous metabolic activation, dichloromethane did not induce gene mutations at the Hprt locus (Jongen et al., 1981).

(v) Cell transformation

Virus-infected Fischer rat and Syrian hamster embryo cells were transformed after treatment with dichloromethane in vitro (Price et al., 1978; Hatch et al., 1982).

(b) Bacterial and other systems

See Table 4.5

Table 4.5. Studies of genotoxicity with dichloromethane in non-mammalian systems in vitro.

Table 4.5

Studies of genotoxicity with dichloromethane in non-mammalian systems in vitro.

Mutagenicity

Gene mutations were induced in Salmonella typhimurium strains TA100, TA1535, and TA98 exposed to dichloromethane vapour in a closed chamber with or without exogenous metabolic activation (JETOC, 1997).

The relationship between the metabolism of dichloromethane and mutagenicity has been examined in several studies with various assays for bacterial mutation. For example, Jongen et al. (1982) showed that while dichloromethane was directly mutagenic in S. typhimurium TA100, mutagenic activity was enhanced by addition of rat liver microsomes or cytosolic fraction (this implicated enhanced metabolism of dichloromethane by CYP and GST, respectively). In contrast, Green (1983) tested the mutagenicity of dichloromethane in the same S. typhimurium strain and observed an increase in mutagenic activity only when rat liver post-mitochondrial S9 fraction was added and not rat liver microsomes.

To further illustrate the complexities of how the two metabolic pathways interact to promote mutagenesis, Dillon and colleagues examined the involvement of endogenous and exogenous GSH using wild-type S. typhimurium TA100 and a GSH-deficient strain (NG54) that contains approximately 25% of the GSH content as the wild-type strain (Dillon et al., 1992). The influence of addition of rat liver S9 fraction, microsomes, or cytosol fractions was also studied. The NG54 strain was slightly less responsive to dichloromethane exposure, addition of rat liver cytosol marginally increased the mutagenic response to dichloromethane, but addition of GSH had little effect (Dillon et al., 1992).

DeMarini and colleagues assessed dichloromethane mutagenicity by using a Salmonella TA1535 strain that had been modified by the cloning of the rat gene for GSTT11 into its genome (DeMarini et al., 1997). This modified strain, called RSJ100, showed a positive mutagenic response to dichloromethane that was predominantly (96–100%) due to mutations that were GC→AT transitions. Interestingly, only 15% of the mutations were GC→AT transitions in the TA100 strain, a homologue strain that lacks the rat GSTT11 gene. These results suggested that different reactive metabolites are formed in the two strains, which leads to different mutations.

Studies using the liquid plate incorporation assay gave negative results (e.g. Zeiger & Dellarco, 1990), with the exception of one study reporting positive results in strain TA1535 transfected with rat Gstt1 (Thier et al., 1993). Dichloromethane also induced mutation in Escherichia coli (Dillon et al., 1992; Zielenska et al., 1993; Graves et al., 1994a; JETOC, 1997) and gene conversion and mutation in Saccharomyces cerevisiae (Callen et al., 1980). In Drosophila melanogaster dichloromethane did not induce sex-linked recessive lethal mutations (Gocke et al., 1981; Kramers et al., 1991).

4.3. Other mechanistic data relevant to carcinogenicity

Few experimental studies have examined the potential for non-genotoxic mechanistic events to play a role in carcinogenesis caused by dichloromethane in tissues that are targets for carcinogenesis in studies in experimental animals. In long-term studies of dichloromethane exposure in mice, elevations in liver-cell proliferation were not observed (Foley et al., 1993; Casanova et al., 1996). In the mouse lung, exposure to dichloromethane results in toxicity to Clara cells, which are secretory cells in the primary bronchioles. Acute exposure to dichloromethane produces vacuolization of Clara cells, which is not sustained with long-term exposure (Foster et al., 1992).

One recent genomics study in vitro compared the effects of dichloromethane and other volatile organic solvents (benzene, toluene, o-xylene, ethylbenzene, and trichloroethylene) on gene expression in human promyelocytoc leukaemia HL-60 cells (Sarma et al., 2010). Equi-toxic concentrations of all solvents were used in studies of gene expression (80% and 50% cell viability). Based on the overall changes in gene expression, dichloromethane exhibited a response that was distinct from other solvents; however, common signatures were identified. These included induction of the immune response, apoptosis, cell cycle regulation, and transport pathways. Select transcripts from these pathways were tested by real-time polymerase chain reaction (PCR) in two other cell lines, human erythromyeloblastoid leukaemia K562 and human leukaemic monocyte lymphoma U937. [The Working Group noted that these data were difficult to interpret as the study appeared not to use proper multiple-testing correction to determine significance of both individual genes and pathways.]

4.4. Organ toxicity

The toxicity of dichloromethane has been reviewed previously (Dhillon & Von Burg, 1995; WHO, 1996; Green, 1997).

4.4.1. Neurotoxicity

(a) Humans

Temporary neurobehavioural effects have been reported (Putz et al., 1979; Winneke, 1981), or not (Gamberale et al., 1975) after exposure to dichloromethane at doses as low as 200 ppm [694 mg/m3]. Cerebral damage after exposure to dichloromethane has been reported (Barrowcliff & Knell, 1979).

(b) Experimental systems

Increase in concentrations of astroglial proteins S-100 and glial fibrillary acidic protein was found in the frontal and sensory motor cerebral cortex of gerbils exposed to dichloromethane at 210 or 350 ppm for 3 months (Rosengren et al., 1986). DNA concentration was also measured as a possible index of astroglial proliferation. DNA concentration was not increased in the frontal and sensory motor cerebral cortex, but was decreased in the hippocampus at 210 and 350 ppm, and in the cerebellar hemispheres (Rosengren et al., 1986).

4.4.2. Liver

(a) Humans

An exposure-related increase in serum bilirubin was observed in workers exposed to dichloromethane, but no other sign of liver injury or haemolysis was reported (Ott et al., 1983)

(b) Experimental systems

A 2-year study of exposure to dichloromethane by inhalation in F344 rats reported that the incidence of some non-neoplastic liver lesions was significantly elevated in response to treatment when compared with concurrent controls (NTP, 1986). These liver lesions were haemosiderosis, focal necrosis, cytoplasmic vacuolization, and bile duct fibrosis in males, and focal granulomatous inflammation, haemosiderosis and cytoplasmic vacuolization in females. In the same study, liver cytological degeneration was observed in female B6C3F1 mice.

A 2-year study of exposure to dichloromethane by inhalation in F344 rats reported that the incidence some non-neoplastic liver lesions (acidophilic, basophilic and vacuolated cell foci in males) was significantly elevated in response to treatment when compared with controls (JISHA, 2000a). In the same study, liver granulation and peripheral vacuolation were observed in male and female BDF1 mice.

Increased liver weight associated with glycogen accumulation in the hepatocytes, but no hepatotoxicity, was observed in another study of carcinogenicity in mice, in which an elevated incidence of hepatic tumours was observed (Kari et al., 1993). An experiment in female B6C3F1 mice showed that the proportion of S-phase cells was frequently higher in altered foci than in cells from the areas of the liver with normal architecture, but similar to that in the altered foci from non-treated mice (Foley et al., 1993). Administration of dichloromethane to B6C3F1 mice by gavage (1000 mg/kg, single dose) or inhalation (4000 ppm [13 900 mg/m3] dichloromethane for 2 hours) did not induce DNA synthesis, as measured by the number of cells in S-phase ([3H]thymidine incorporation) (Lefevre & Ashby, 1989). When female B6C3F1 mice were exposed to dichloromethane at 1000, 2000, 4000, or 8000 ppm [3470, 6940, 13 900 or 27 800 mg/m3] for 6 hours per day, 5 days per week, for up to 4 weeks, followed by a recovery period of 1–2 weeks (Foley et al., 1993), the hepatocyte labelling index was mostly decreased. There were, however, transient increases in the labelling index in the groups at 4000 and 8000 ppm at 2 weeks and in the group at 1000 ppm at 1 week.

In Sprague-Dawley rats, two doses of dichloromethane at 1250 mg/kg given by gavage for 4 and 21 hours, there was no effect on serum alanine aminotransferase levels, or hepatic GSH or CYP content, but hepatic ornithine decarboxylase activity increased in 3 out of 15 rats (Kitchin & Brown, 1989).

Hepatotoxic effects were seen after exposure to near-lethal concentrations of dichloromethane in mice (Gehring, 1968). Continuous exposure of mice to dichloromethane at 5000 ppm [17 400 mg/m3] by inhalation caused swelling of the rough endoplasmic reticulum, fatty changes in the liver, and necrosis of individual hepatocytes (Weinstein et al., 1972). Slight liver damage was also observed after administration of dichloromethane (133–665 mg/kg bw) by gavage in mice (Condie et al., 1983).

Exposure of guinea-pigs to dichloromethane at 5200 ppm [18 000 mg/m3] by inhalation for 6 hours increased hepatic concentrations of triglyceride (Morris et al., 1979). Exposure of guinea-pigs to dichloromethane at approximately 11 000 ppm [38 200 mg/m3] for 6 hours also increased hepatic concentrations of triglyceride, but concomitant exposure to ethanol at 21 400–24 100 ppm [40 200–45 300 mg/m3] blocked this effect (Balmer et al., 1976).

4.4.3. Cardiovascular system

(a) Humans

Of four epidemiological studies on mortality from cardiovascular disease, two studies showed increased mortality from ischaemic heart disease in workers exposed to dichloromethane, compared with an internal reference group or a non-exposed cohort, although mortality did not increase compared with the general population (Tomenson et al., 1997; Tomenson, 2011).

(b) Experimental systems

No data were available to the Working Group.

4.4.4. Respiratory system

(a) Humans

No data were available to the Working Group.

(b) Experimental systems

Nasal cavity lesions of olfactory epithelium and hyperplasia of the terminal bronchiole have been reported in male and female BDF1 mice in a 2-year study of exposure to dichloromethane by inhalation (JISHA, 2000b). The incidence of eosinophilic changes in the respiratory epithelium was also elevated in female mice in this study.

F344/N rats were exposed to dichoromethane at a concentration of 0, 1000, 2000, or 4000 ppm by inhalation for 6 hours per day, 5 days per week, for 102 weeks. Squamous metaplasia of the nasal cavity was observed as a treatment-related non-neoplastic change in rats (Mennear et al., 1988).

The labelling index in bronchiolar epithelium (in two branches proximal to the terminal bronchiole and in the terminal bronchioles themselves) in female B6C3F1 mice exposed to dichloromethane at 2000 ppm for 2–26 weeks decreased to 40–60% of the value for control mice. Exposure to dichloromethane at 8000 ppm led to a smaller decrease in labelling index. No pathological changes were found in the exposed lungs (Kanno et al., 1993). In male B6C3F1 mice exposed to dichloromethane by inhalation (6 hours, single dose), vacuolation of bronchiolar cells was observed at exposure levels ≥ 2000 ppm [6940 mg/m3], while no effect was observed at levels ≤ 1000 ppm [3470 mg/m3] (Foster et al., 1994). Pretreatment with the CYP inhibitor piperonyl butoxide (300 mg/kg, administered intraperitoneally) 1 hour before exposure abolished the toxic effect in bronchiolar cells, while buthionine sulfoximine (1 g/kg, administered intraperitoneally), which decreased the pulmonary GSH content by 50%, had no protective effect. In Clara cells isolated after exposure to dichloromethane (≥ 1000 ppm), the proportion of cells in S-phase was increased.

4.4.5. Kidney

(a) Humans

No data were available to the Working Group.

(b) Experimental systems

In a 2-year study in female F344 rats exposed to dichloromethane by inhalation, kidney tubular degeneration was reported to be significantly elevated in response to treatment when compared with controls (NTP, 1986). In the same study, kidney tubule casts were observed in male and female B6C3F1 mice.

In a 2-year study in female F344 rats exposed to dichloromethane by inhalation, the incidence of chronic nephropathy was significantly elevated in response to treatment when compared with controls (JISHA, 2000a). In a study in similarly exposed BDF1 mice, basophilic change, lymphocytic infiltration and proximal tubule vacuolation were observed (JISHA, 2000b).

After intraperitoneal administration of dichloromethane at near-lethal doses, hydropic degeneration was observed in the mouse kidney (Klaassen & Plaa, 1966), no kidney damage was observed after administration of dichloromethane at doses of 133–665 mg/kg bw by gavage (Condie et al., 1983). Slight calcification of the renal tubules in mongrel dogs was seen after intraperitoneal administration of dichloromethane at near-lethal doses (Klaassen & Plaa, 1967).

In rats, intraperitoneal administration of dichloromethane at 1330 mg/kg bw produced renal proximal tubular swelling (Kluwe et al., 1982). After a similar dose administered by gavage, a transient elevation in blood urea nitrogen levels and decreased urine output, coinciding with cloudy swelling of tubular cells, were observed (Marzotko & Pankow, 1988). Urinary flow was already decreased at the lowest dose tested (3.1 mmol/kg bw; 263 mg/kg bw). In F344/N rats exposed to dichloromethane at 0, 1000, 2000, or 4000 ppm by inhalation, for 6 hours per day, 5 days per week, for 102 weeks, treatment-related degeneration of kidney tubules was reported (Mennear et al., 1988).

4.4.6. Spleen

(a) Humans

No data were available to the Working Group.

(b) Experimental systems

In F344/N rats were exposed by inhalation to dichloromethane at 0, 1000, 2000, or 4000 ppm, for 6 hours per day, 5 days per week, for 102 weeks, fibrosis of the spleen was observed as a treatment-related non-neoplastic change (Mennear et al., 1988).

4.5. Susceptible populations

4.5.1. Polymorphisms

(a) CYP2E1

The association between exposure to organic solvents including dichloromethane and NHL was investigated in relation to different genetic variations in four metabolic genes – CYP2E1, microsomal epoxide hydrolase (EPHX1), myeloperoxidase (MPO), and quinone oxidoreductase (NQO1) – using unconditional logistic regression models based on data collected from women in Connecticut, USA, in 1996–2000 (Barry et al., 2011). Overall associations between total NHL and dichloromethane (OR, 1.69; 95% CI, 1.06–2.69), carbon tetrachloride (OR, 2.33; 95% CI, 1.23–4.40), and methyl chloride (OR, 1.44; 95% CI, 0.94–2.20) were increased among women of genotype TT for rs2070673 in the CYP2E1 gene (dichloromethane: OR, 4.42; 95% CI, 2.03–9.62; P interaction < 0.01; carbon tetrachloride: OR, 5.08; 95% CI, 1.82–14.15; P interaction = 0.04; and methyl chloride: OR, 2.37; 95% CI, 1.24–4.51; P interaction = 0.03). In contrast, no effects of these solvents were observed among women of genotype TA/AA. Similar patterns were observed for dichloromethane and diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma (Barry et al., 2011). [The Working Group noted that the functional significance of this polymorphism was unknown.]

(b) GSTT1

GSTT1 polymorphisms may result in interindividual variation in the ability to metabolize dichloromethane by GSH conjugation; some individuals (non-conjugators) completely lack GSH conjugation activity. Because GSH conjugation of dichloromethane leads to formation of reactive and genotoxic metabolites, it is plausible that diminished or lack of GSH conjugation activity will lead to reduced risk of carcinogenesis. For instance, in the absence of GSTT1, exposure to dichloromethane did not lead to formaldehyde production in human erythrocytes (Hallier et al., 1994), and DNA–protein-cross-links were not detected in human liver cells (Casanova et al., 1997). This could be relevant to multiple target tissues that express GSTs, including the liver, kidney, brain, and lung (Sherratt et al., 1997, 2002).

Interindividual variation in the conjugation of dichloromethane with GSH by cytosolic GST in vitro was investigated in 22 samples of human liver (Bogaards et al., 1993). In nine of the liver samples, the α-, mu-, and pi-class GST subunits were quantified. In two of these samples, no activity was observed towards dichloromethane, while α-, mu-, and pi-class subunits were expressed in these human liver cytosolic samples, suggesting no relationship between enzymatic activities and dichloromethane with these classes of GST.

Hallier et al. (1993) found that dichloromethane induced sister-chromatid exchange in the human lymphocytes of non-conjugators donors lacking GST activity, but not in those of conjugators. However, Olvera-Bello et al. (2010) demonstrated that the group with high GSTT1 activity showed a larger increase in the frequency of sister-chromatid exchange induced by dichloromethane than did the groups with low and medium GSTT1 activity.

Garte et al. (2001) showed major and significant differences in the allele and genotypes frequencies between ethnic groups, especially between Asians and Caucasians (Table 4.6).

Table 4.6. Frequencies of GSTT1*0 gene polymorphism in Caucasians and Asians.

Table 4.6

Frequencies of GSTT1*0 gene polymorphism in Caucasians and Asians.

4.5.2. Life stage

Few studies have examined the influence of life stage on dichloromethane-induced toxicity or carcinogenesis. Most of the available studies related to potential differences in toxicokinetics across life stages, with no chemical-specific data on toxicodynamic differences. With respect to absorption and distribution, no age-dependent differences in the partition coefficient for mixtures of volatile organic solvents have been observed in rats (Mahle et al., 2007). No data on life-stage–dependent differences in elimination or excretion were available.

Although no direct data on life-stage–dependent differences in dichloromethane metabolism were available, based on information on the ontogeny of CYP2E1 and GSTT1, such differences are plausible. In humans, CYP2E1 activity is low during gestation and the early neonatal period (Choudhary et al., 2005), but no data were available on the ontogeny of GSTT1. Data in experimental animals suggested that both CYP2E1 (Choudhary et al., 2005) and GST (Cui et al., 2010) expression are low during gestation, and peak between 0 and 12 days after birth. Czekaj et al. (2010) found that CYP2E1 expression increases further in older adult rats. Although the qualitative patterns were similar, the available data were insufficient to estimate the magnitude of any differences in the proportion of oxidative metabolism versus conjugation during early life stages as compared with during adulthood. Therefore, there was inadequate evidence to conclude whether there are differences in susceptibility as a function of life stage as a result of changes in metabolism.

4.6. Mechanistic considerations

See Table 4.7

Table 4.7. Relationship between the glutathione/glutathione S-transferase (GSH/GST) pathway and dichloromethane-induced genotoxicity.

Table 4.7

Relationship between the glutathione/glutathione S-transferase (GSH/GST) pathway and dichloromethane-induced genotoxicity.

Two important metabolic pathways for the metabolism of dichloromethane have been characterized in humans and experimental animals. One pathway is CYP2E1-mediated reductive dehalogenation, which ultimately generates CO and CO2 as stable end products. One of the intermediates, formyl chloride, can react with nucleophiles. GSH conjugation, catalysed primarily by GSTT1, is the other important metabolic pathway of dichloromethane, resulting in the formation of reactive metabolites, including formaldehyde and S-chloromethyl GSH.

Supporting evidence for the GST pathway include in-vitro studies from human-derived tissue or cells, in-vivo studies in rodents, in-vitro studies in rodent-derived tissue or cells, in-vitro mutagenicity studies in microorganisms, and biochemical studies with purified enzymes. Humans are polymorphic for GSTT1, with a proportion of the population showing no activity towards dichloromethane. CYP2E1 catalytic activity predominates at relatively low concentrations of substrate, but there is ample evidence that GST-mediated metabolism eventually predominates at higher concentrations (Gargas et al., 1986; Clewell, 1995; Bos et al., 2006). Such higher concentrations of dichloromethane are readily observed in occupational settings and in some environmental exposures. Moreover, with continued exposure to dichloromethane, even at relatively low concentrations, CYP2E1 readily becomes saturated. Overall, evidence strongly supports qualitative similarities in both oxidative and GST-mediated metabolism of dichloromethane between humans and rodents.

Differences in activity levels and tissue and cellular distributions of GSTT1 exist across species. For instance, in the liver and lung, two sites where tumours are observed in mice in long-term bioassays (NTP, 1986), GSTT1 activity was greater in mice than in rats or humans (Reitz et al., 1989; Thier et al., 1998). Humans, however, have GSTT1 activity in erythrocytes that is comparable to that in the mouse liver, while neither rats nor mice exhibit GSTT1 activity in erythrocytes (Thier et al., 1998). Additionally, in the mouse liver, nuclear localization of GSTT1 was observed in hepatocytes, while in the human liver, nuclear localization of GSTT1 was observed in bile-duct epithelial cells (Quondamatteo et al., 1998; Sherratt et al., 2002). Thus, while the metabolic pathways are similar across species, the target tissues and cell types of GSTT1 metabolism differ across species.

Dichloromethane has been evaluated for genotoxicity in several test systems, both in the presence or absence of metabolic activation. In human cell lines or isolated cells, dichloromethane has been reported to induce micronucleus formation and sister-chromatid exchange (Hallier et al., 1993; Doherty et al., 1996; Olvera-Bello et al., 2010); but studies of DNA–protein cross-links, DNA single-strand binding proteins (SSBs), and unscheduled DNA synthesis have largely given negative results (Jongen et al., 1981; Graves et al., 1995; Casanova et al., 1997). In one study, the extent of sister-chromatid exchange was greater in cells from individuals without GST activity (Hallier et al., 1993). In another study, by contrast, the extent of sister-chromatid exchange was greater in cells from individuals with high GSTT1 activity (Olvera-Bello et al., 2010). In experimental animals, dichloromethane-induced genotoxicity also tended to correlate with GST activity, with positive results in cells derived from mouse liver and lung, which also exhibited the greatest GST activity (Graves et al., 1994b, 1995; Casanova et al., 1997). Similarly, after exposure to dichloromethane in vivo, although many studies gave negative results for genotoxicity, positive results in multiple measures of genotoxicity were reported in tissues with GST-mediated metabolism, such as the mouse liver and lung (Allen et al., 1990; Casanova et al., 1992, 1996; Graves et al., 1995; Sasaki et al., 1998). Finally, several studies in non-mammalian in-vitro systems showed evidence for mutagenicity, particularly in systems in which GST activity is present or exogenously enhanced (Jongen et al., 1978, 1982; Gocke et al., 1981; Green, 1983; Thier et al., 1993; DeMarini et al., 1997; Pegram et al., 1997). Overall, genotoxicity attributable to dichloromethane appears to be strongly associated with GST-mediated metabolism, consistent with the formation of reactive metabolites through this pathway. However, in two available studies in human cells, enhanced genotoxicity was observed without GSTT1 activity in one, and with high GSTT1 activity in another.

Increased liver weights and glycogen deposition were observed after long-term exposure to dichloromethane, but their relationship to carcinogenesis was not clear (NTP 1986; Kari et al., 1993). Several studies in mice have shown that liver cell proliferation does not increase with exposure to dichloromethane, suggesting that proliferation does not play a role in hepatocarcinogenesis in the mouse (Lefevre & Ashby, 1989; Foley et al., 1993; Casanova et al., 1996). In the mouse lung, acute exposure to dichloromethane leads to vacuolization of Clara cells, but this effect appears to be transient (Foster et al., 1992), so is unlikely to be involved in carcinogenesis in the mouse lung. Mice exposed to dichloromethane for up to 26 weeks had no pathological changes in the lung, but exhibited a decrease in cell proliferation in this tissue. Neurological, renal, spleen, reproductive, and developmental toxicity have also been reported in humans or experimental animals, confirming the widespread distribution of dichloromethane or its metabolites.

Together, the relationship between GSTT1-mediated metabolism, formation of reactive metabolites, the association between GST activity and genotoxicity, and the presence of GSTT1 polymorphisms in the human population suggest that GSTT1 polymorphism may lead to differential susceptibility to dichloromethane-related carcinogenesis. However, no studies have directly investigated whether an association exists between GSTT1 polymorphism and the incidence of cancer. One study has reported an association between a CYP2E1 polymorphism and NHL in dichloromethane-exposed individuals (Barry et al., 2011). Whether this is due to differences in formation of CYP2E1-mediated metabolites, which may also be reactive, or to a shift in the proportion of GST-mediated reactive metabolites is unknown.

© International Agency for Research on Cancer, 2017. For more information contact publications@iarc.fr.
Bookshelf ID: NBK436271

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