MAY/ JUNE 1993 VOLUME 6, NUMBER 3 0 Copyright 1993 by the American Chemical Society
Invited Review Metabolism of N,N-Dimethylformamide: Key to the Understanding of Its Toxicity Andreas Gescher Mechanisms of Drug Toxicity Research Group, Pharmaceutical Sciences Institute, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. Received January 4, 1993
Introduction NJV-Dimethylformamide [DMF,' (CH&NCHO, Chart I] is a common polar solvent that has a worldwide annual production of about 2 X lo5tons (1)and is used in a wide variety of industrial processes, among them the manufacture of synthetic fibers, leathers, films, and surface coatings. DMF possesses a number of intriguing biological properties, the most prominent of which is its ability to induce the terminal differentiation of certain malignant cells (2). Gastric irritation and hepatotoxicity are the major untoward effects which have been reported in workers exposed to DMF (for review see ref 3). Recently, more than half of the workforce in an industrial environment in which they were occupationally exposed to DMF under conditions of poor industrial hygiene showed elevated indices of hepatotoxicity (4, 5). These reports were considered of sufficient gravity to warrant the publication of a reminder of methods of protection to be used to avert adverse health effects of DMF by the US. National Institute for Occupational Safety and Health (NIOSH) (6). The toxicity of DMF in animals has recently been cogently reviewed (7). DMF has been implicated as the cause of alcohol incompatibility reactions in exposed workers (8-1 1 ) and is suspected of being a carcinogen (12, 'Abbreviations: AMCC, N-acetyl-S-(N-methylcarbamoy1)cysteine; AUC, area under the plasma formamide concentration curve; DMF, NJVdimethylformamide; HMMF, N-(hydroxymethy1)-N-methylformamide; NMF, N-methylformamide; P450 2E1,cytochrome P450 isozyme also known as CYP2E1, IIE1, ac, j, or 3a; SMG, S-(N-methylcarbamoy1)glutathione.
Chart I. Structures of DMF, NMF, Their Metabolites, and N-Methyl-N-hydroxyformamidea
DMF
N-(Hydroxymethy1)lormamide
0 H\
It
N - C -SG CHj
HMMF
NMF
Formamide
N-Hydroxy-N-meihylformamide NHCOCHa
0
H\ N-C
It
- S-
CHj
SMG
I
CH2-CH
I
COOH
AMCC
SG = glutathionyl.
13). However, the results of an extensive retrospective study on workers who had been potentially exposed to DMF for many years suggest that the carcinogenic risk associated with occupational exposure is very low (14,15). Over the last decade, a considerable amount of data has been accumulated on the xenobiochemistry of formamides, especially of N-methylformamide (NMF,' CH3NHCHO), the N-demethyl analogue of DMF which has undergone clinical evaluation as an anticancer agent (16-20). This work indicates strongly that the hepatotoxicity of formamides is intrinsically linked to their metabolism. The implications of this suggestion for DMF are discussed here
0S93-228~/93/2706-0245$04.00/0 0 1993 American Chemical Society
246
Chem. Res. Toxicol., Vol. 6, No. 3, 1993
with the intent of integrating the current knowledge of this class of compound. Thus this review should help establish the basis for understanding the mechanism of DMF toxicity. Furthermore, it is hoped that it will aid the formulation of hypotheses which when tested might provide answers to the few questions still open pertaining to the way in which this agent causes unwanted effects.
Metabolism of DMF Primary Metabolites of DMF. The major pathway of metabolic disposition of DMF involves the hydroxylation of its methyl moieties. The primary product of this metabolic route is N-(hydroxymethy1)-N-methylformamide [HMMF, CH3(CHzOH)NCHOI. HMMF, in turn, can decompose to NMF chemically with concomitant elimination of formaldehyde at a rate which depends on the pH and temperature of the environment. It is almost twenty years since NMF was first characterized by GLC as the major metabolite of DMF in animals and humans in vivo (21,22).However, more recent investigations have demonstrated that the DMF metabolite excreted in the urine is not NMF but its precursor HMMF (23,24). Only traces of NMF were detected in the urine of animals which had received DMF (25). The reason for the discrepancy between the old and the recent reports is a consequence of the fact that HMMF is unstable to GLC conditions and undergoes thermolytic degradation to NMF on the column (23,24). In aqueous solution of neutral or mildly acidic pH, HMMF is relatively stable. Analysis of urine samples using 'H-NMR spectroscopy proved that the metabolite in the urine which gave NMF on GLC analysis was actually HMMF (25). HMMF is the major urinary excretion product of DMF in humans. Volunteers who were exposed for 8 h to 30 mg.m-3 DMF vapor excreted only 0.3% of the absorbed dose as DMF, but 22.3% as HMMF (26). The possibility that NMF could be present, under certain conditions, as a metabolite in addition to HMMF has been difficult to assess because of the instability of HMMF to GLC analysis. In a recent investigation of the plasma disposition of DMF and its metabolites in rats and mice which had inhaled DMF vapor, discrimination between NMF and HMMF was achieved (27). NMF constituted between 30% and 60 % of HMMF plasma levels, depending on exposure and sampling time. In an accompanying study NMF was similarly found together with HMMF in the blood of monkeys (28). Consistent with the earlier observations, only minute concentrations of NMF were observed in the urine in these studies. The notion that cytochromeP450 mediates the oxidation of DMF to HMMF was already suspected in the original investigations of DMF metabolism (21,22,29).Recently, compelling evidence has been presented which suggests a crucial role for the P450 enzyme P450 2E1 (30). This conclusion is based on the findings, among others, that pretreatment of rats with the P450 2E1 inducer, acetone, increased the rate at which liver microsomes generated HMMF from DMF, that diethyldithiocarbamate inhibited it, and that a polyclonal antibody against rat P450 2E1 decreased the rate of this biotransformation dramatically in rat and human liver microsomes. The generation of HMMF from DMF by rat liver microsomes was found to be subject to a primary deuterium kinetic isotope effect (30). Comparison of the metabolism of DMF with that of hexadeuteriomethyl-DMF [(C2H3)2CHOlyielded the in-
Gescher
termolecular kinetic isotope effect, while comparison of the rate of oxidation of the two isotopomeric methyl moieties in trideuteriomethyl-DMF [CH3(C2H3)NCH0] afforded a value of 5.2 for the intramolecular kinetic isotope effect. Primary deuterium kinetic isotope effects of similar magnitude have been found for the metabolic N-dealkylation of other NJV-dialkylamidesand are thought to imply that the mechanism of oxidation involves the abstraction of a hydrogen atom from the methyl group as opposed to abstraction of electrons from the nitrogen as the initiating event (31). Secondary Metabolite of DMF. HMMF and NMF, once generated from DMF, undergo further metabolism. Formamide (H2NCHO) is a product of this metabolism, and it was found by GLC analysis in the urine of rodents and humans after exposure to DMF (21,22). This species may well be the product of the thermal decomposition of N-(hydroxymethy1)formamide [(CH20H)NHCHOl generated by enzymatic N-methyl oxidation of NMF. Degradation of N - (hydroxymethy1)formamideto formamide would be analogous to the thermal degradation of HMMF to NMF. In the pharmacokinetic study on human volunteers who inhaled 30 mg.m-3DMF vapor for 8 h (vide supra) 13.2% of the dose appeared in the urine as formamide (26). An alternative pathway for metabolism of NMF and HMMF involves oxidation of the formyl group. The metabolic end product of this metabolic avenue is N-acetylS-(N-methylcarbamoy1)cysteine(AMCC),lwhich has been characterized as a major urinary metabolite of DMF in rodents and humans (32). Human volunteers who were exposed to 30 mg.m-3 DMF vapor for 8 h excreted 13.4% of the dose as AMCC (26). AMCC is also the most important metabolite of NMF (32-34). The oxidation of the formyl moiety in the NMF molecule furnishes a reactive intermediate, an event which has been demonstrated to occur in vivo (35) and in liver microsomes (36) and hepatocytes in vitro (37). In view of its short-lived nature, it is not surprising that the chemical structure of the metabolic intermediate has not yet been resolved. Methyl isocyanate (CH3NCO) is a chemically feasible candidate. N-Methylcarbamic acid (CHzNHCOOH) is another possibility, although an anhydride such as N-methylcarbamoyl phosphate (CH3NHCOOP03-H)would be chemically more plausible. The intermediate reacts spontaneously with GSH to yield S-(N-methylcarbamoy1)glutathione(SMG),l which is ultimately excreted as the mercapturate AMCC. SMG has been detected in the bile of rodents after application of NMF (35)or DMF2 and in incubations of NMF with liver microsomes in the presence of GSH (36). Authentic HMMF, like NMF, generates SMG in incubations with liver microsomes and GSH. Since the carbinolamide itself is a substrate for the microsomal enzymes which catalyze the reaction (30,361,the oxidation can occur at the formyl carbon of HMMF. That is, HMMF does not have to be converted to NMF first for the reaction to proceed. Nevertheless, the amount of SMG generated from HMMF in vitro was much smaller than that produced from NMF under identical conditions (30). Therefore, it seems unlikely that direct oxidation of HMMF contributes substantially to the formation of SMG from DMF in vivo. The metabolism of SMG generated in vivo to AMCC involvespresumably formation and subsequent acetylation 'R. Hyland, P. Davis, and T. A. Baillie, unpublished.
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 247
Invited Reviews
Scheme I. Metabolic Generation of the Reactive Intermediate, Probably Methyl Isocyanate, from
NMF
N-C H3C’
\H
of S-(N-methylcarbamoy1)cysteineas is the general fate of GSH conjugates. The pivotal oxidation step in the generation of SMG from NMF is catalyzed by P450 2E1 (38), as is the metabolic oxidation of DMF to HMMF. The involvement of P450 2E1 in this biotransformation is borne out by the observations, among others, that inducers of the enzyme increased the rate of microsomal SMG generation and that it was inhibited efficiently by an antibody against rat P450 2E1. It has been argued that it is the formyl moiety of the NMF molecule which is the site of metabolic oxidation en route to the reactive intermediate (35).From a mechanistic standpoint, it is conceivable that the initial oxidative attack occurs at the nitrogen atom instead, yielding a hydroxamic acid which could subsequently eliminate water to give the reactive intermediate (Scheme I). The formation of AMCC from NMF was found to be subject to a primary deuterium kinetic isotope effect of 4.5 when the rate of AMMC formation in vivo was compared between NMF and its isotopomer deuterioformyl-NMF (CH3NHC2HO)(35),pointing to the cleavage of the formyl C-H bond as the rate-determining step in the overall reaction. As the hydroxamic acid lacks hepatotoxic properties and as it has never been identified as a product of NMF, presumably it does not play a role as intermediate metabolite of NMF.
Mechanism of Toxicity of DMF What is the evidence for the contention that metabolites are responsible for the toxicity of DMF, and which metabolites have been incriminated as toxic species? The original studies on DMF metabolism related its adverse effects to its biotransformation to NMF (21, 22, 29). Consistent with this notion, monoalkylformamides such as NMF are clearly more potently toxic than DMF (3,391. The difference in hepatotoxic potential between DMF and NMF is illustrated by experiments in BALB/c mice in which a single dose ip of 3.4 mmol kg-l NMF caused hepatotoxicity, whereas DMF at up to 41 mmol k g l did not elicit any manifestation of liver damage (40). Similarly, in suspensions of isolated hepatocytes in vitro, in which 1-5 mM NMF caused irreparable damage, DMF at concentrations of up to 10 mM was innocuous (37). Furthermore, HMMF is much less toxic than NMF (41). Understanding of the mechanism by which DMF causes toxicity was aided considerably by the characterization of AMCC as a major metabolite of DMF (32). AMMC has been identified in rats as a metabolite of methyl isocyanate,
the chemical which caused the Bhopal diaster (42). Thus these findings can be integrated into a scheme in which the reactive intermediate of DMF, presumably methyl isocyanate, generates the toxic lesion, unless it undergoes rapid detoxification via trapping by thiol-containing molecules to form N-methylcarbamic acid thioesters such as SMG. The main support for this hypothesis is provided by studies on a range of amides which demonstrated that hepatotoxic potential is restricted to N-alkylformamides and closely associated with their propensity to undergo metabolism to N-alkylcarbamic acid thioesters (40). Among these compounds NMF and its homolog N-ethylformamide yielded considerable amounts of thiocarbamate in vivo and were also found to be the most hepatotoxic members of the series. Formation of SMG was originally thought to be an event which brings about the detoxification of the reactive product of NMF oxidation (35). Consistent with this idea are the findings that depletion of GSH by inhibition of its synthesis using buthionine sulfoximine exacerbated the toxicity of NMF, whereas administration of N-acetylcysteine protected against NMF-induced hepatotoxicity (43). In addition, a study of the chemical and toxicological properties of N-alkylcarbamic acid thioesters showed that they also possess cytotoxic properties (44). This finding is probably related to their ability to transfer the N-alkylcarbamoyl moieties to bionucleophiles, for example, cysteine, GSH, or thiol-containing protein, in in vitro incubations (45). Therefore, it appears that the reaction of the primary product of formamide formyl oxidation with GSH has to be considered as a partial detoxification step to species which retain a degree of cytotoxicity. As indicated above, it is not easy to elicit DMF-induced hepatotoxicity in rodents, and exceptionally high doses appear to be required. Therefore, the effect of modulation of thiol status on the toxicity of DMF remains to be elucidated. Likewise, studies of the binding of reactive metabolites derived from P4C1-labeledsubstrate to tissue macromolecules are needed to define more exactly the metabolic toxification of DMF. Analogous studies on NMF have helped substantially to unravel the relationship between hepatic metabolism and toxicity (46). Details of the cascade of metabolites and products of chemical breakdown which precede the generation of AMCC from DMF have not yet been fully explored. As they bear upon the interpretation of the role of metabolism in the mechanism by which DMF may cause its toxicity, they are worthy of discussion. In light of the findings summarized above it is likely that the formation of the mercapturate from DMF requires the intermediacy of NMF. The metabolic pathway leading from DMF to the toxic intermediate and then to AMCC can be rationalized in either of two ways (Scheme 11). First, two separate pathways have been postulated for DMF: One involves a detoxification step, leading to HMMF as an innocuous excretion product, and the route of activation yielding NMF as the major product (pathway A in Scheme 11) (27). Second, HMMF may be the direct precursor of NMF and thus play the role of the pivotal intermediate and proximate toxicant (pathway B, Scheme 11). The latter hypothesis, which postulates HMMF as the direct precursor of NMF and thus of the toxic intermediate and of SMG, is eminently plausible on chemical grounds. As HMMF is relatively stable in biological media, the formation of NMF from HMMF, both of which have been
248 Chem. Res. Toxicol., Vol. 6, No. 3, 1993
Gescher
Scheme I1
AHMMF
A.
GSH
B. DMF
P450 2E1
HMMF
HCHO
SMG
--
AMCC
found in the blood after exposure to DMF, may require catalysis by enzymes. However, the suggestion that HMMF is a proximate toxicant and the direct progenitor of NMF is difficult to reconcile with the fact that the carbinolamide itself is devoid of marked toxic properties on ip injection in rodents. One would expect HMMF to exert considerable toxicity in vivo if it were enzymically converted to NMF, which is very hepatotoxic. It could be argued that HMMF, which is a very hydrophilic substance, does not enter hepatocytes readily when adminstered to animals via the ip route, and thus it may hardly undergo metabolism under these conditions. In contrast, when formed metabolically within the hepatocyte, it may be available for conversion to NMF. Obviously, the current state of knowledge does not permit an unambiguous judgment as to which of these two hypotheses is correct. Clearly, the mechanistic details of the metabolic process which links DMF with NMF remain a fertile ground for further discovery.
Pharmacokinetics of Excretion of DMF Metabolites The emerging details of the mechanism of DMF metabolism help us explain certain characteristics of the pharmacokinetic behavior of DMF and its metabolites. In humans exposed to DMF vapors only a minute portion of absorbed DMF is excreted unchanged in the urine, and it is dose-dependent, consistent with extensive metabolism (22,26). Mass balance studies on the fate of DMF have not been conducted in humans. Therefore, it is unclear whether a fraction of the absorbed DMF is exhaled as DMF or NMF. Elimination of HMMF is rapid; consequently, it does not accumulate in the body. This conclusion was supported experimentally in several studies on volunteers who were exposed repeatedly to airborne DMF vapor at concentrations of 26 mg-m-3 during 6 h daily for 5 days (47),30 mgm-3 during 8 h daily for 5 days (26),or 63 mgm-3 during 4 h daily for 5 days (22). Even though HMMF undergoes oxidation in hepatic microsomes to a minor extent (vide supra), it is predominantly excreted unaltered when administered as such. In the urine of rats which received authentic HMMF, approximately 60 5% of the dose could be recovered unchanged (48). Consistent with these observations, the plasma elimination half-life of HMMF (10 mg/kg, ip) in rats was 3 h, thus only a fourth of that observed for the same dose of NMF.3 In contrast G. L. Kennedy and S. G. Hundley, unpublished.
to the rapid urinary elimination of HMMF, the DMF metabolite AMCC is excreted unusually slowly. This observation was made in humans after exposure to DMF vapor (26)and in rodents after DMF administration via the ip route (49). The urinary excretion curves in humans for DMF, HMMF, and AMCC differ dramatically. In the 8-h DMF exposure study peak levels were achieved 6-8 h after the start of the exposure in the case of DMF and HMMF, but not before 24-36 h for AMCC (26). The decreasing portion of the urine concentration time curves for DMF, HMMF, and AMCC in this study afforded excretion half-lives of approximately 2, 4, and 23 h, respectively. In view of these findings it is not surprising that repeated inhalation of DMF for 5 days caused the accumulation of AMCC (26). What are the reasons for the delay in the excretion of AMCC and for its slow excretion half-life? Authentic AMCC ingested by a volunteer was excreted rapidly and without delay (26). So the explanation does not appear to involve the disposition of AMCC. Instead, inhibition of its metabolic generation might be the cause for the delayed appearance with the urine. The metabolic oxidations leading to formation of HMMF from DMF and SMG from NMF are mediated mainly or perhaps exclusively by the same enzyme, namely, P450 2E1 (30,38).In a recent kinetic investigation, the affinity of DMF for the microsomal enzyme was shown to be an order of magnitude higher than that of NMF (30). These studies revealed that the difference between DMF and NMF in the apparent K , value obtained with rat liver microsomes, 0.2 mM for the former and 4.3 mM for the latter, renders DMF an efficient competitive inhibitor of the generation of the reactive intermediate, and thus SMG, from NMF. This finding means that in vivo SMG formation, and, consequently, excretion of AMCC, is retarded as long as blood and tissue levels of DMF are high. The ability to inhibit its own P450 2El-medited metabolism also explains findings in rats in vivo reported by Lundberg et al(50,51). In this study, DMF decreased the severity and postponed the onset of NMF-induced hepatotoxicity as measured by elevated serum sorbitol dehydrogenase levels. Likewise, the administration of 480 mg kg’ DMF resulted in the delayed start of toxicity compared to that triggered by 240 mg k g l . The corollary of the ability of DMF to inhibit its own metabolism to NMF is the possibility that manifestations of detrimental effects of DMF on the health of workers after overexposure may develop well beyond the end of the work shift. The slow urinary excretion of AMCC is probably due to its ability to undergo transcarbamoylation reactions (26). The reactive precursor of AMCC and SMG, presumably methyl isocyanate, cannot only be trapped by GSH, but it is also bound to cellular proteins. Methyl isocyanate bound as thiocarbamate can be released as the reaction is reversible. That thiocarbamates such as SMG and AMCC can donate their N-alkylcarbamoyl moieties to nucleophilic cell constituents was shown by Pearson et al. (45). The “removal” of methyl isocyanate from protein is followed by a sequence of rapid processes such as conjugation with GSH, metabolism to AMCC, and renal elimination of AMCC. The release of “bound” methyl isocyanate from cellular proteins might be the slowest step in the overall transformation of DMF to AMCC and thus be at the root of the slow excretion of AMCC.
Invited Reviews
Species Differences in DMF Metabolism The association of the toxicity of formamides with their metabolism, especially the biotransformation pathway which leads ultimately to AMCC, implies that it is pivotal to elucidate differences between animals and humans in the metabolism of DMF. A better understanding of such differences might contribute to the choice of a suitable animal model in experiments designed to help with the assessment of the risk to humans exposed to DMF. With this rationale in mind, the excretion into the urine of DMF, HMMF, formamide, and AMCC after exposure to DMF was compared between mice, rats, hamsters, and humans (49). Rodents received DMF at three dose levels (0.1,0.7, and 7 mmol kg') via ip injection, and the yield of metabolites for the lowest dose are quoted below as percentage. Humans inhaled 60 mgm-3 DMF vapor for 8 h and absorbed 49.3 pmol kgl. All species disposed of DMF via the same metabolic routes. However, there were considerable quantitative differences. DMF was hardly detectable. The amount of HMMF varied between 8.4% of the dose of DMF in mice and 36.8 % in rats, as compared to 25.9% in humans. Similarly, the portion of the dose eliminated as formamide fluctuated between 22.9 % (hamster) and 37.5% (rat) and was 14.2% in humans. In contrast, AMCC contributed only between 1.6% and 5.2% of the dose excreted in the urine of rodents, whereas in the 8 humans the amount of AMCC which was measured in the urine varied between 10% and 23%, with a mean of 14.5 7%. This study seems to suggest that humans may be exposed to larger amounts of the reactive metabolite of DMF than rodents. Therefore, the authors speculated that humans may be more sensitive to adverse effects induced by DMF than are rodents (49). The reason for the discrepancy between rodents and humans in AMCC generation is unclear. It is unlikely that differences in P450 2E1 are involved, as such differences should also cause a parallel divergence in HMMF excretion, which was not observed. Furthermore, the K , and V,,, values established for the oxidation of NMF to the reactive intermediate (trapped as SMG) in liver microsomes from rat and human were very similar to each other (30). The possibility has to be borne in mind that SMG or an SMG metabolite might be excreted via the feces. This hypothesis has hitherto not been tested. Fecal excretion of such DMF metabolites might contribute to the overall amount of conjugation product of reative intermediate which is discharged from the body, and this amount may not correspond with the quantity of AMCC eliminated via the urine. In this case the conclusion drawn from the observation of the difference between species in urinary excretion of AMMC (49)would not be valid. In adifferent study, DMF, HMMF, and NMF were detected in the blood of monkeys (28)and mice and rats (27) after exposure to DMF, and there was some difference in metabolite concentration between species. The values for the area under the plasma concentration versus time curve (AUC) (expressed as pgh mL-') for DMF and for the sum of HMMF and NMF measured within 24 h after a 6-h exposure to either 250 ppm (mice and rats) or 100 ppm DMF (monkeys) were as follows: DMF, 330 in mice, 800 in rats, and 29 in male and 43 in female monkeys; HMMF plus NMF, 528 in mice, 1010 in rats, and 231 in male and 274 in female monkeys (27,28). These data are indicative of greater systemic exposure to DMF in rodents compared to monkeys at comparable inhalation concentrations.
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 249
Furthermore, the systemic exposure to DMF as reflected by DMF plasma levels in these studies was disproportionately elevated with increasing concentration of DMF vapors in the air, indicating saturation. AUC values for DMF increased by 19- to 37-fold in male and by 35- to 54-fold in female monkeys as the concentration of airborne DMF increased from 100 to 500 ppm (28); a similar elevation was observed in rats and mice (27). I t is worth noting that in rodents DMF toxicity, where it has been observed, seemed to manifest itself only at exposure levels which apparently saturate metabolism (7). In contrast, monkeys exposed to up to 500 ppm DMF for 90 days did not exhibit any sign of toxicty (52). In view of the fact that monkeys are, on the whole, better predictors of chemical hazards in humans than are rodents, this finding suggests that the risk associated with exposure to humans is indeed minimal. As SMG in the plasma or AMCC in the urine was not measured in this study, it is unknown whether and how DMF-derived thiocarbamate levels differed between monkeys and rodents. In view of the lack of knowledge with respect to the mass balance of administered DMF, activities of DMF-metabolizing enzymes in the different species, and the consequence of route for administration on metabolite excretion, a conclusive verdict on the relative susceptibilities of different species toward DMF-induced toxicity is tenuous.
Conclusions The studies described above provide many of the components of the jigsaw puzzle which, when assembled, might furnish, albeit still incompletely, the picture describing the role of metabolism in the hepatic toxicity caused by DMF. Efficient metabolic oxidation to HMMF is the quantitatively most important biotransformation route of DMF, and it occurs in all species,including human. Extensive studies of the metabolism and toxicity of NMF would suggest that the generation of NMF as proximate toxicant from DMF is an obligatory step in the events which determine DMF toxicity. How NMF is produced from HMMF in vivo, perhaps via enzyme catalysis, is still speculative. NMF, but also to a minor extent HMMF, undergoes oxidation in the formyl moiety to generate a reactive, probably the ultimate, toxic metabolite. This compound is perhaps methyl isocyanate. NMF, once formed from DMF, is either reabsorbed or biotransformed further so efficiently that hardly any NMF appears with the urine. The elusive metabolic intermediate might never be unambiguously identified, but without doubt it reacts avidly with GSH. Whether the intermediate and/or its conjugation product SMG carbamoylates cellular targets specifically as a fateful prelude to hepatocytotoxicity is as yet unknown. P450 2E1 plays clearly an important role in the catalysis of the metabolic events leading from DMF to SMG. This fact implies that modulation of levels of this enzyme by dietary constituents, for example, ethanol, might alter the susceptibility of humans toward the detrimental effects of DMF. Ethanol is a competitive inhibitor of P450 2E1, and its chronic consumption leads to P450 2E1 induction (53). Interactions between DMF and ethanol have indeed been demonstrated in humans. Exposure to DMF after drinking ethanol decreased blood concentrations of HMMF (and/or NMF), elevated urinary DMF concentrations (54), and retarded the appearance of HMMF and AMCC in the urine (26).
250 Chem. Res. Toxicol., Vol. 6, No. 3, 1993
Metabolites of DMF are perhaps responsible for the alcohol incompatibility reaction observed sporadically after exposure of workers to DMF. It is unlikely that DMF itself acts like disulfiram, as it does not itself inhibit alcohol or aldehyde dehydrogenase enzymes in vitro (55, 56). Whether metabolites of DMF can inhibit the enzymes which rid the organism of acetaldehyde, the hangoverproducing ethanol metabolite, remains to be elucidated. SMG, AMCC, and its cysteine precursor might be the culprits, as there is a striking similarity in structure between these thioesters and disulfiram and dioxiram, powerful inhibitors of aldehyde dehydrogenase (57). Disulfiram and dioxiram cause enzyme inhibition via thiocarbamoylation or carbamoylation of the thiol moiety at the active site, and it is conceivable that the DMF metabolites N-methylcarbamoylate this site. Most of the experiments reviewed above which address the association between formamide metabolism and toxicity have dealt with the liver-damaging properties of this class of compound. It is therefore pertinent to stress that the role which HMMF, NMF, the reactive intermediate, and/or SMG play in the mechanisms by which DMF elicits toxic manifestations other than hepatotoxicity is unclear. Biological monitoring of occupational exposure to DMF is currently based on the determination in urine samples of the metabolite which affords NMF on GLC analysis and which is predominantly HMMF, as outlined above. On the basis of the evidence summarized above, AMCC might perhaps merit consideration as a suitable biomarker for the assessment of individual sensitivity to DMFinduced adverse effects on the liver. The main reason for this proposal is the fact that the health risk associated with exposure to DMF seems to be linked to the metabolic pathway leading to this metabolite, whereas the role of HMMF has still not been fully clarified. In a recent study of the percutaneous absorption of DMF in humans the suitability of HMMF and AMCC as biomarkers was compared (58). The authors concluded that AMCC would indeed reflect the integral exposure to DMF more reliably than HMMF, regardless of route of absorption or withinday variation. Before the ultimate verdict is passed on the case for the use of AMMC as biomarker instead of, or in addition to, HMMF, a study should be conducted in which the total yield and chemical identity of all metabolites of DMF are accounted for in all excreta, not only in the urine. DMF is a small and chemically rather simple molecule, and only a decade and a half ago one would not have predicted the intricacy of its metabolic fate. The knowledge of its metabolism will eventually help with formulation of guidelines applied to the handling of this and related compounds. Therefore, DMF could be looked at as a paradigm for those chemicals in the human environment the metabolism of which is still unknown, but might be crucial for the explanation of their biological properties.
Acknowledgment. I thank Drs. M. D. Threadgill, J. M r b , and T. A. Baillie for their collaboration and ideas, the many postdoctoral fellows and graduate students involved with the work on the formamides for their motivation and enthusiasm, the Cancer Research Campaign, the Health and Safety Executive and the Medical Research Council for grant support, and Drs. G. L. Kennedy and S. G. Hundley of Dupont-Haskell Laboratories for help with assembling this review.
Gescher
References (1) Eberling, C. L. (1980) Dimethylformamide.
In Kirk-Othmer Encyclopaedia of Chemical Technology (Mark, H. F., Othmer, D. F., Overberger, C. G., and Seaborg, G. T., Eds.) pp 263-268, Wiley and Sons, New York. (2) Dexter, D. L. (1979) N,N-Dimethylformamide-induced morphological differentiation and reduction of tumorigenicity in cultured mouse rhabdomyosarcoma cells. Cancer Res. 37, 3136-3140. (3) Gescher, A. (1990) Dimethylformamide. In Ethel Browning’s Toxicity andMetabolism oflndustrial Soluents (Buhler, D. R., and Reed, D. J., Eds.) Vol. 2, pp 149-159, Elsevier, Amsterdam. (4) Redlich, C. A., Beckett, W. S., Sparer, J., Barwick, K. W., Riely, C. A., Miller, H., Sigal, S.L., Shalat, S. L., and Cullen, M. R. (1988) Liver disease associated with occupational exposure to the solvent dimethylformamide. Ann. Int. Med. 108, 680-686. (5) Wang, J. D., Lai, M. Y., Chem, J. S., Lin, J. M., Chiang, J. R., Shiau, S.J., and Chang, W. S.(1991) Dimethylformamide-induced liver damage among synthetic leather workers. Arch. Enuiron. Health 46, 161-166. (6) National Institute for Occupational Safety and Health, Cincinnati, OH (1991) Preventing adverse health effects from exposure to dimethylformamide (DMF). Am. Ind. Hyg. Assoc. J . 52, A160A162. (7) Kennedy, G. L. (1986) Biological effects of acetamide, formamide and their monomethyl and dimethyl derivatives. Crit. Reu. Toxicol. 17, 129-182. (8) Chivers, C. P. (1978) Disulfiram effect from inhalation of dimethylformamide. Lancet 8059, 331. (9) Lyle, W. H., Spence, T. W. M., McKinneley, W. M., and Duckers, K. (1979) Dimethylformamide and alcohol intolerance. Br. J. Ind. Med. 36, 63-66. (10) Tomasini, M., Todaro, A., Piazzoni, M., and Peruzzo, G. F. (1983) Pathology of dimethylformamide: Observations on 14 cases. Med. Lou. 74, 217-220. (11) Cox, N. H., and Mustchin, C. P. (1991) Prolonged spontaneous and alcohol-induced flushing due to the solvent dimethylformamide. Contact Dermatitis 24, 69-70. (12) Ducatman, A. M., Conwill, D. E., and Crawl, J. (1986) Germ cell tumors of the testicle among aircraft repairmen. J. Urol. 136,834836. (13) Levin,S. M.,Baker,D.B., Landrigan, P. J.,Monaghan,S. V.,Frumin, E., Braithwaite, M.,and Towne, W. (1987)Testicularcancer inleather tanners exposed to dimethylformamide. Lancet 8568, 1153. (14) Chen, J. L., Fayerweather, W. E., and Pell, S.(1988) Cancer incidence of workers exposed to dimethylformamide and/or acrylonitrile. J. Occup. M e d . 30, 813-818. (15) Walrath, J. (1989) A case-controlled study of cancer among Du Pont employees with potential for exposure to dimethylformamide. J. Occup. Med. 31, 432-438. (16) Laird Myers, W. P., Karnofsky, D. A., and Burchenal, Y. H. (1956) The hepatotoxicity of N-methylformamide in man. Cancer 9,945954. (17) McVie, J. G., Ten Bokkel Huinink, W. W., Simonetti, G., and Dubbelman, R. (1984) Phase I trial of N-methylformamide. Cancer Treat. Rep. 69, 607-610. (18) Ettinger, D. S., Orr, D. W., Rice, A. P., and Donehower, R. C. (1985) Phase I study of N-methylformamide in patients with advanced cancer. Cancer Treat. Rep. 69, 489-493. (19) Eisenhauer, E. A., Weinerman, B. H., Kerr, I., and Quirt, I. (1986) Toxicity of oral N-methylformamide in three phase I1 trials: A report from the National Cancer Institute of Canada Trials Group. Cancer Treat. Rep. 70, 881-883. (20) Rowinsky, E. K., Grochow, L. B., Hantel, A., Ettinger, D. S., Vito, B. L., and Donehower, R. C. (1989) Assessment of N-methylformamide (NMF) administered orally on a three times weekly schedule: a phase I study. Inuest. New Drugs 7, 317-325. (21) Kimmerle, G., and Eben, A. (1975) Metabolism studies of N,Ndimethylformamide. I. Studies in rata and dogs. Int. Arch. Arbeitsmed. 34, 109-126. (22) Kimmerle, G., and Eben, A. (1975) Metabolism studies of N,Ndimethylformamide. 11. Studies in persons. Int. Arch. Arbeitsmed. 34, 127-136. (23) Brindley, C., Gescher, A,, and Ross, D. (1983) Studies of the metabolism of dimethylformamide in mice. (!hem.-Biol. Interact. 45, 387-392. (24) Scailteur, V., De Hoffmann, E., Buchet, J. P., Lauwerys, R. (1984) Study on in vivo and in uitro metabolism of dimethylformamide in male and female rats. Toxicology 29, 221-234. (25) Kestell, P., Gill, M. H., Threadgill, M. D., Gescher, A., Howarth, 0. W., and Curzon, E. H. (1986) The formation and metabolism of N-hydroxymethyl compounds. 7. Identification by proton NMR of N-(hydroxymethy1)-N-methylformamideas the major urinary metabolite of N,N-dimethylformamide in mice. Life Sci. 38, 719724.
Invited Reviews (26) MrAz, J., and Nohova, H. (1992) Absorption, metabolism and elimination of N,N-dimethylformamide in humans. Int. Arch. Occup. Enuiron. Health 64, 85-92. (27) Hundley, S. G., Lieder, P. H., Valentine, R., Maalley, L. A., and Kennedy, G. L. Dimethylformamide pharmacokinetics following inhalation exposure to rats and mice. Drug Chem. Toxicol. (in press). (28) Hundley, S. G., McCooey, K. T., Lieder, P. H., Hurtt, M. E., and Kennedy, G. L. Dimethylformamide pharmacokinetics following inhalation exposure in monkeys. Drug Chem. Toxicol. (in press). (29) Barnes, J. R., and Ranta, K. E. (1972) Metabolism of dimethylformamide and dimethylacetamide. Toxicol. Appl. Pharmacol. 23, 271-276. (30) Mrlz, J., Jheeta, P., Gescher, A., Hyland, R., Thummel, K., and Threadgill, M. D. (1993) Investigation of the mechanistic basis of N,N-dimethylformamide toxicity. Metabolism of N,N-dimethylformamide and its deuterated isotopomers by cytochrome P450 2E1. Chem. Res. Toxicol. 6, 197-207. (31) Hall, L. R., and Hanzlik, R. P. (1990) Kinetic deuterium isotope effects on the N-dimethylation of tertiary amides by cytochrome P-450. J . B i d . Chem. 265, 12349-12355. (32) Mrlz, J., and Turecek, F. (1987) Identification of N-acetyl-S-(Nmethylcarbamoyl)cysteine, a human metabolite of N,N-dimethylformamide and N-methylformamide. J . Chromatogr. Biomed. Appl. 414, 399-404. (33) Kestell, P., Gledhill, A. P., Threadgill, M. D., and Gescher, a. (1986) S-(N-Methylcarbamoy1)-N-acetylcysteine: A urinary metabolite of the hepatotoxic experimental antitumour agent N-methylformamide (NSC3051) in mouse, rat and man. Biochem. Pharmacol. 35,22832286. (34) Tulip, K., Timbrell, J. A., Nicholson, J. K., Wilson, I., and Troke, J. (1986) A proton magnetic resonance study of the metabolism of N-methylformamide in the rat. Drug Metab. Dispos. 14,746-749. (35) Threadgill, M. D., Axworthy, D. B., Baillie, T. A., Farmer, P. B., Farrow, K. C., Gescher, A., Kestell, P., Pearson, P. G., and Shaw, A. J. (1987) Metabolism of N-methylformamide in mice: Primary kinetic deuterium isotope effect and identification of S-(N-methylcarbamoy1)glutathione as a metabolite. J . Pharmacol. E x p . Ther. 242, 312-319. (36) Cross, H., Dayal, R., Hyland, R., and Gescher, A. (1990) N-Alkylformamides are metabolized to N-alkylcarbamoylating species by hepatic microsomes from rodents and humans. Chem.Res. Toxicol. 3, 357-362. (37) Shaw, A. J., Gescher, A., and Mrlz, J. (1988) Cytotoxicity and metabolism of the hepatotoxin N-methylformamide and related formamides in mouse hepatocytes. Toxicol. Appl. Pharmacol. 95, 162-170. (38) Hyland,R., Gescher, A.,Thu"el,K.,Schiller, C., Jheeta,P., Mynett, K., Smith, A. W., and Mrlz, J. (1992) Metabolic oxidation and toxification of N-methylformamide catalyzed by the cytochrome P450 isoenzyme CYP2E1. Mol. Pharmacol. 41, 259-266. (39) Gescher, A. (1990) N-Methylformamide. In Ethel Browning's Toxicity and Metabolism oflndustrial Soluents (Buhler, D. R., and Reed, D. J., Eds.) Vol. 2, pp 169-177, Elsevier, Amsterdam. (40) Kestell, P., Threadgill, M. D., Gescher, A., Gledhill, A. P., Shaw, A. J., and Farmer, P. B. (1987) An investigation of the relationship between hepatotoxicity and the metabolism of N-alkylformamides. J . Pharmacol. Exp. Ther. 240, 265-270. (41) Gate,E. N.,Threadgill,M. D., Stevens,M. F. G.,Chubb, D.,Vickers, L. M., Langdon, S. P., Hickman, J. A., and Gescher, A. (1986) Structural studies on bioactive compounds. 4. A structureantitumor activity study on analogues of N-methylformamide. J . Med. Chem. 29, 1046-1052.
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 251 (42) Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D. H., Grillo, M. P., and Baillie, T. A. (1990) S-(N-Methylcarbamoy1)glutathione: a reactive S-linked metabolite of methyl isocyanate. Biochem. Biophys. Res. Commun. 166, 245-250. (43) Pearson, P. G.,Gescher, A., and Harpur, E. S. (1987) Hepatotoxicity of N-methylformamide in mice. I. Relationship to glutathione status. Biochem. Pharmacol. 36, 381-384. (44) Han, D. H., Pearson, P. G., Baillie, T. A., Dayal, R., Tsang, L. H., and Gescher, A. (1990) Chemical synthesis and cytotoxic properties of N-alkylcarbamic acid thioesters, metabolites of hepatotoxic formamides. Chem. Res. Toxicol. 3, 118-124. (45) Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D.-H., andBaillie, T. A. (1991) Carbamoylation of peptides and proteins in vitro by S-(N-methy1carbamoyl)glutathioneand S-(N-methylcarbamoy1)cysteine, two electrophilic S-linked conjugates of methyl isocyanate. Chem. Res. Toxicol. 4,436-444. (46) Pearson, P. G., Gescher, A., and Harpur, E. S. (1987) Hepatotoxicity of N-methylformamide in mice. 2. Covalent binding of metabolites of ['4C]-labelled N-methylformamide. Biochem. Pharmacol. 36,385390. (47) Krivanek, N. D., McLaughlin, M., and Fayerweather, W. E. (1978) Monomethylformamide levels in human urine after repetitive exposure to dimethylformamide vapor. J. Occup. Med. 20, 179182. (48) Scailteur, V., and Lauwerys, R. (1984) In vivo metabolism of dimethvlformamide and relationshk to toxicitv in the male rat. Arch. T"oxico1. 56, 87-91. (49) Mrlz, J.. Cross, H., Gescher. A., Threadgill, M. D.. and Flek, J. (1989) Differences between humans and rodents in the metabolic toxification of N,N-dimethylformamide. Toxicol. Appl. Pharmacol. 98, 507-516. (50) Lundberg,I.,Lundberg, S.,andKronevi,T. (1981)Someobservations on dimethylformamide hepatotoxicity. Toxicology 22, 1-7. (51) Lundberg, I., Pehrsson, A., Lundberg, S., Kronevi, T., and Lidum, V. (1983) Delayed dimethylformamide biotransformation after high exposures in rats. Toxicol. Lett. 17, 29-34. (52) Hurtt, M. E., Placke, M. E., Killinger, J. M., Singer, A. W., and Kennedy, G. L. (1992) 13-Week inhalation toxicity study of dimethylformamide (DMF) in cynomolgusmonkeys. Fundam. Appl. Toxicol. 18, 549-601. (53) Johansson, I., Ekstrom, G., Scholte, B., Puzycki, D., Jornvall, H., and Ingelman-Sundberg, M. (1988) Ethanol-, fasting- and acetoneinducible cytochromes P-450 in the rat liver: Regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry 27, 1925-1934. (54) Eben, A., and Kimmerle, G. (1976) Metabolism studies of N,Ndimethylformamide 111. Studies about the influence of ethanol in persons and laboratory animals. Int. Arch. Occup. Enuiron. Health 36, 243-265. (55) Sharkawi, M. (1979) Inhibition of alcohol dehydrogenases by dimethylformamide and dimethylsulfoxide. Toxicol. Lett. 4,493497. (56) Elovaara, E., Marselos, M., and Vainio, H. (1983) N,N-Dimethylformamide-induced effects on hepatic and renal xenobiotic enzymes with emphasis on aldehyde metabolism in the rat. Acta Pharmacol. T O X ~ 53, C O159-165. ~. (57) Kitson, T. M. (1991) Effect of some thiocarbamate compounds on aldehyde dehydrogenase and implications for the disulfiram ethanol reaction. Biochem. J. 278, 189-192. (58) Mrhz, J., and Nohova, H. (1992) Percutaneous absorption of N,Ndimethylformamide in humans. Int. Arch. Occup. Enuiron. Health 64, 79-83.
