formic acid excretion in rats exposed to trichloroethylene: a possible explanation for renal...

Toxicology 127 (1998) 39–47

Formic acid excretion in rats exposed to trichloroethylene: apossible explanation for renal toxicity in long-term studies

Trevor Green *, Jacky Dow, John R. Foster, Paul M. Hext

Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, UK

Received 8 October 1997; accepted 29 January 1998

Abstract

Rats exposed to trichloroethylene, either by gavage or by inhalation, excreted large amounts of formic acid in urinewhich was accompanied by a change in urinary pH, increased excretion of ammonia, and slight increases in theexcretion of calcium. Following a single 6-h exposure to 500 ppm trichloroethylene, the excretion of formic acid wascomparable to that seen after a 500 mg/kg dose of formic acid itself, yet the half-life was markedly different. Formateexcretion in trichloroethylene treated rats reached a maximum on day 2 and had a half-life of 4–5 days, whereasurinary excretion was complete within 24 h following a single dose of formic acid itself. Formic acid was shown notto be a metabolite of trichloroethylene. When rats were exposed to 250 or 500 ppm trichloroethylene, 6 h/day, for 28days, the only significant effects were increased formic acid and ammonia excretion, and a change in urinary pH.There was no evidence of morphological liver or kidney damage. Long-term exposure to formic acid is known tocause kidney damage suggesting that excretion of this acid may contribute to the kidney damage seen in the long-termstudies with trichloroethylene. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Trichloroethylene; Renal toxicity; Formic acid

1. Introduction

Trichloroethylene has been shown to benephrotoxic in animals, particularly the rat, fol-lowing long-term exposure either by gavage or

inhalation. In a number of studies, the nephrotox-icity in male rats has been accompanied by smallincreases in the incidences of kidney tubular celladenomas and adenocarcinomas (NTP, 1983,1988, 1990; Maltoni et al., 1986, 1988). Thesetumours have not been seen in the absence ofkidney toxicity suggesting that toxicity is a pre-requisite for the development of these tumours.

* Corresponding author. Tel.: +44 162 5515458; fax: +44162 5586396.

0300-483X/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.

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T. Green et al. / Toxicology 127 (1998) 39–4740

A number of studies have investigated themechanism(s) involved in the development ofboth the nephrotoxicity and the low incidences ofkidney tumours seen in male rats exposed totrichloroethylene. These tumours were not seen infemale rats or mice exposed under the same con-ditions. A minor metabolic pathway involvingconjugation of trichloroethylene with glutathionewas identified several years ago and has beenlinked with the adverse renal toxicology seen inthe long-term studies (Dekant et al., 1986, 1990;Commandeur and Vermeulen, 1990; Birner et al.,1993; Goeptar et al., 1995; Henschler et al., 1980,1995; Birnauer et al., 1996; Green et al., 1997). Invivo, this pathway occurs at very low levels in allspecies; typically the major urinary metabolite,N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-acetyl DCVC), accounts for approximately0.005% of the administered dose oftrichloroethylene (Commandeur and Vermeulen,1990; Birner et al., 1993; Birnauer et al., 1996;Green et al., 1997). The precursor to the majorurinary metabolite, S-(1,2-dichlorovinyl)-L-cys-teine (DCVC), is known to be activated by therenal enzyme b-lyase to reactive species which arenephrotoxic in rodents and mutagenic in bacteria(Green and Odum, 1985), and consequently itsformation is suggested to be the basis of theeffects seen in the long-term studies.

Quantitation of the pathway leading to DCVCin rats and mice failed to find the expected corre-lation between metabolism by this pathway andthe species difference in renal cancer seen in thebioassays. The levels of N-acetyl DCVC in mouseurine were several fold higher than those in the rat(Birner et al., 1993) and the mouse was signifi-cantly more susceptible to the acute nephrotoxiceffects of DCVC than the rat (Eyre et al., 1995a,b;Green et al., 1997). The potency of DCVC as arenal carcinogen has also been questioned. Thelevels of this metabolite formed fromtrichloroethylene are approximately three ordersof magnitude lower than the acute renal NOELfor DCVC in the rat (Green et al., 1997), and inthe long-term, DCVC itself failed to cause renalcancer in rats, again at dose levels which wereseveral orders of magnitude higher than the levelsof DCVC found in rats dosed with

trichloroethylene (Terracini and Parker, 1965).Thus, in addition to the lack of species specificity,there is a question about the toxicological signifi-cance of the very low levels of DCVC formedfrom trichloroethylene.

The lack of mechanistic data to support theassumption that the nephrotoxicity and low inci-dence of rat kidney tumours are a result of themetabolism of trichloroethylene to DCVCprompted the present search for an alternativeexplanation. In this paper, the acute renal toxicityof trichloroethylene has been assessed over a 28-day period, and the novel finding that rats ex-posed to this chemical excrete large amounts offormic acid is reported.

2. Materials and methods

2.1. Chemicals

Trichloroethylene with a minimum purity of\99% was supplied by BDH (Poole, Dorset,UK). All other chemicals were of analytical gradeor the highest grade available. [1,2-14C]-1,1,2-Trichloroethylene (14C-trichloroethylene) was sup-plied in sealed glass ampoules by CambridgeResearch Biochemicals (Northwich, Cheshire)(19.3 mCi/mmol; 97%).

2.2. Animals

Male Fischer 344 rats (180–220 g bodyweight)were supplied by Harlan Olac UK. The animalswere group housed in stainless steel cages in tem-perature controlled rooms equipped with a 12-hlight/dark cycle. Food (CTR diet, Special DietServices Ltd, Witham, Essex, UK) and water wereprovided ad libitum.

2.3. In 6i6o studies

2.3.1. Acute studiesRats (n=3) were given a single gavage dose of

14C-trichloroethylene (1000 mg/kg; 52 mCi/kg).Further groups of rats (n=5 per group) weregiven either five consecutive daily gavage doses oftrichloroethylene (1000 mg/kg in corn oil, 5 ml/

T. Green et al. / Toxicology 127 (1998) 39–47 41

kg) or exposed to 500 ppm by inhalation, whole-body in glass chambers (Jencons Metabowls, Jen-cons, Leighton Buzzard, UK), for 6 h. In eachstudy, urine was collected over dry ice for 24 hprior to dosing and at 24-h intervals for up to 5days after dosing. In a further study rats (n=3)were each given a single gavage dose of formicacid (500 mg/kg as sodium formate in water, 5ml/kg) and urine collected over dry ice at 24-hintervals for 2 days.

Urine samples were analysed for formic acid byNMR (Bruker 400 or 500 MHz spectrometer) orusing a modification of the biochemical method ofGrady and Osterloh (1986), (the pH of the bufferused in the assay was change to pH 7.6 and thereaction time was increased to 60 min). Radioac-tivity in the urine of rats dosed with 14C-trichloroethylene was determined by liquidscintillation counting (Packard 2500TR).Trichloroethanol glucuronide was assayed inurine by first hydrolysing the conjugate to freetrichloroethanol. Urine samples (0.1 ml) were ad-justed to pH 6.8 by the addition of 0.15 ml 0.1 Mpotassium phosphate buffer and incubated at37°C for 2 h with 100 units of b-glucuronidase(Type VII-A from E. coli ; Sigma-Aldrich, UK).The urine was then extracted twice with 4 ml ofdiethyl ether. An aliquot of the ether extract(typically 0.05 ml) was methylated with an ethe-real solution of diazomethane and analysed bygas chromatography using a Hewlett Packard5890 chromatograph fitted with an electron cap-ture detector. A 1.5 m×2 mm I.D. Carbopak C0.1% SP1000 column was used at 135°C. Theretention time of the methyl ester oftrichloroethanol was 5.5 min. Standard curvesprepared from urine samples containing knownamounts of trichloroethanol were analysed usingthe same procedure. Calcium and ammonia wereanalysed by Scientific Analysis Laboratories(Manchester, UK). Urinary pH was alsodetermined.

2.3.2. 28-Day studyGroups of rats (n=5) were exposed to 0, 250

and 500 ppm trichloroethylene, 6 h/day, for 1, 7,15, 21 and 28 days. At the end of each exposureperiod, urine was collected over dry ice for 18 h

prior to the rats being killed by exsanguinationunder terminal anaesthesia (Fluothane, ZenecaPharmaceuticals, Macclesfield, Cheshire, UK).Livers and kidneys were removed, weighed andtaken for histopathological examination. Portionsof liver from left, median and right lobes and onewhole kidney were fixed in 10% (w/v) neutralbuffered formol saline, dehydrated through anascending ethanol series and embedded in paraffinwax. Sections (5-mm) were cut and stained withhaematoxylin and eosin. Kidney sections werealso stained for calcium. Blood samples were cen-trifuged to separate plasma, and alkaline phos-phatase (ALP), alanine transaminase (ALT),aspartate transaminase (AST), gamma-glutamyltranspeptidase (GGT), urea (BUN) and creatininewere determined by standard automated methods.Formic acid in plasma was quantified by NMR.Urine samples were analysed for creatinine(CRT), protein (TP), ALP, N-acetyl glu-cosaminidase (NAG), GGT, calcium, ammoniaand formic acid. Urinary pH was determined.

3. Results

3.1. Acute studies

Rats dosed with trichloroethylene, either bygavage or by inhalation, were found to haveexceptionally high levels of formic acid in theirurine. In contrast, the urine samples collectedfrom rats prior to dosing or from control animalshad little or no formic acid (Fig. 1). The excretionof formic acid following a single 1000 mg/kggavage dose was higher on day 2 (5.892.1 mg/24h) than day 1 (4.191.3 mg/24 h). Similarly, fol-lowing a single exposure to 500 ppmtrichloroethylene, excretion of formic acid washighest on day 2 and continued for at least 5 dayswith a half-life of between 4 and 5 days (Fig. 2).The same trend was also seen in the plasma levelsof formic acid (Fig. 2). This was in marked con-trast to that following a single oral dose of formicacid itself when urinary excretion (18.5797.10mg formic acid; 20% of the dose) was completewithin 24 h of dosing. The concentrations offormic acid in urine during daily gavage dosing of


Fig. 1. NMR spectra of urine from an untreated rat and from a rat exposed to 500 ppm trichloroethylene for 6 h. Urine wascollected for 18 h post-exposure.


Fig. 2. Formic acid levels in rat urine (shaded) and plasma(hatched) for 5 days following a single 6-h exposure to 500ppm trichloroethylene. Urinary pH is also shown for the sameperiod.

acid would have accounted for up to 21% of thedose. Since the total urinary excretion of radioac-tivity was only 15% of the dose (mainly astrichloroethanol glucuronide) it was concludedthat formic acid was not a metabolite oftrichloroethylene.

3.2. 28-Day study

Livers and kidneys taken throughout the 28-day study were morphologically normal as werethe biochemical parameters of kidney damagewhich were measured in blood and urine (Table1). Plasma creatinine, GGT, ALT, AST, ALP andliver and kidney bodyweight ratios were also un-changed (data not shown). Thus, based on theseassessments, trichloroethylene was neither hepato-toxic nor nephrotoxic following exposure for a28-day period to the dose levels used in the cancerbioassays.

As had been seen following acute exposures,urinary and plasma levels of formic acid weremarkedly increased on day one of the 28-daystudy and remained constant throughout thestudy (Fig. 4). Urinary pH was significantly de-pressed throughout the study (Fig. 4). There wasno evidence of a dose response for any of theseparameters. Calcium levels in urine were not sig-nificantly elevated during the study, althoughthere did appear to be a trend towards increasedcalcium excretion in the exposed groups (Fig. 5).Urinary ammonia excretion was significantly in-creased at the 500 ppm dose level throughout thestudy (Fig. 5). Ammonia was not measured at thelower dose level.

4. Discussion

Trichloroethylene is an extremely weak acutenephrotoxin as exemplified by the present 28-daystudy. There were no morphological changes inthe kidneys at any of the time points, nor werethere any changes in the sensitive biochemicalmarkers normally used to detect kidney damage.Similar findings have been reported following gav-age administration of high dose levels oftrichloroethylene (Stott et al., 1982; Goldsworthy

trichloroethylene (1000 mg/kg) increased over thefirst 4 days to a level (Fig. 3) which was compara-ble to that seen on day 2 following a single 6-hinhalation exposure to 500 ppm (Fig. 2).

The pH of urine collected fromtrichloroethylene dosed rats reflected the excretionof formic acid. Following a single exposure to 500ppm urinary pH was reduced until formic acidexcretion diminished on day 4 when the pH re-turned to that of control urine (Fig. 2).

In rats dosed with 1000 mg/kg of 14C-trichloroethylene, 14% of the dose was excreted inurine within the first 24 h and 1% on the secondday. 86% of the radioactivity in urine was presentas trichloroethanol glucuronide. Over the same2-day period, up to 13.2 mg of formic acid wereexcreted in urine, the greater amount on the sec-ond day. On a molar basis this amount of formic

Fig. 3. Formic acid levels in rat urine during repeated dailygavage dosing of 1000 mg/kg trichloroethylene.


Table 1Biochemical markers of kidney damage in blood and urine from rats exposed to 0, 250 and 500 ppm trichloroethylene, 6 h/day forup to 28 days

Dose (ppm) No. of expo- TP (mg/dl) GGT (IU/l) ALP (IU/l) CRT (mmol/l) NAG (IU/l) BUN (mmol/l)sures

0 2439251 5229113 83945 6.691.1 6.792.1 ND199951 4959178 1069537 6.091.1 5.491.1 6.290.8

15 238952 4719131 76923 6.191.5 4.290.7 7.091.220694221 4789193 89975 6.191.1 3.590.7 6.990.8193943 398988 6993028 6.390.5 4.291.3 7.591.1

1250 279946 3909241 109971 6.590.3 7.391.5 ND241926 5279146 104948 6.790.77 8.290.7 6.290.5263947 5889208 11194315 5.890.3 5.791.2 6.790.9

21 280982 5479208 103957 6.691.4 5.792.1 5.790.8200958 3059111 77919 5.191.628 3.990.9 6.590.8247941 561987 759141 6.590.7500 7.091.1 ND218918 5279101 106941 5.790.57 6.790.9 5.990.3252921 647980 13395315 5.790.6 4.391.4 5.691.0

21 218945 6329210 73919 6.290.8 4.191.1 6.391.0240955 4379129 7593928 5.991.3 5.091.4 7.290.6

Urine: TP, total protein; GGT, gamma-glutamyl transpeptidase; ALP, alkaline phosphatase; CRT, creatinine; NAG, N-acetylglucosaminidase.Blood: BUN, blood urea nitrogen.

et al., 1988; Green et al., 1997). It would appeartherefore that the nephrotoxicity seen at the samedose levels of trichloroethylene in the chronicstudies is the result of a low level, but sustainedpertubation of the kidney, rather than a frankacute cytotoxicity. Under these circumstances,identifying the cause of the chronic nephrotoxicityseen in the lifetime bioassays is difficult and re-quires a sensitive means of detecting adversechanges in the kidney. Nuclear magnetic reso-nance (NMR) is a technique which has been usedin the past to detect changes in urinary compo-nents which are related to chemically inducedkidney damage (Nicholson and Gartland, 1987;Holmes et al., 1990). The use of NMR for thispurpose initiated the present study when a NMRspectrum of urine from rats dosed withtrichloroethylene revealed a very intense signal at8.5 ppm which was not present in control urine(Fig. 1). The proton resonance of formic acid (8.5ppm) is relatively unique, but its identity wasfurther confirmed by comparison with authenticmaterial, by spiking the urine sample with formicacid and showing an increase in the signal inten-sity, and finally, with an totally independent bio-

chemical assay. Analysis of urine samples fromthese studies and from other rats in this labora-tory has shown that significant levels of formicacid are not excreted in control animals.

The source of the formic acid in urine is atpresent unclear, and is the subject of furtherinvestigation. It appears not to be a metabolite oftrichloroethylene, since neither the rate of excre-tion nor the amounts excreted were compatiblewith the known metabolism of this chemical.Quantitatively, the amount of formic acid in urineexceeded the total amount of trichloroethylenemetabolites. One of the most interesting observa-tions in this study is the slow excretion of formicacid and its very long half-life. Following a singledose of formic acid itself, urinary excretion iscomplete within 24 h, and the half-life in plasmahas been reported to be a short as 12 min (Mal-orny, 1969). In marked contrast, following expo-sure to trichloroethylene the excretion of formicacid is greater on day 2 than on day 1 andcontinues for at least 5 days after dosing. Thisobservation further confirms that formic acid isnot a metabolite since the major metabolites oftrichloroethylene are almost entirely cleared


within 24 h of dosing (Prout et al., 1985). Theprolonged half-life does suggest that either thesynthesis of formic acid is markedly stimulated, orthat clearance is blocked. Formic acid is producedmainly from the catabolism of amino acids and isalmost entirely utilised through tetrahydrofolateor cleared by catalase/hydrogen peroxide to car-

Fig. 5. The excretion of calcium and ammonia in urine fromrats exposed to 0 (shaded), 250 (wide hatched) and 500 (nar-row hatched) ppm (calcium) or 0 and 500 ppm (ammonia)trichloroethylene, 6 h/day for up to 28 days. * PB0.05;** PB0.005.

Fig. 4. Formic acid levels in urine and plasma during a studyin which rats were exposed to 0 (shaded), 250 (wide hatched)and 500 (narrow hatched) ppm trichloroethylene, 6 h/day forup to 28 days. Urinary pH is shown over the same period.

bon dioxide. Incorporation into tetrahydrofolateis the major route in the rat; only about 25% isestimated to be metabolised by catalase. It seemsunlikely that formic acid synthesis is stimulatedfor this duration, and to a level that can no longerbe cleared by these two pathways, and more likelythat trichloroethylene or one of its metabolites insome way interferes with formic acid clearance.This, as yet, undefined mechanism would appearto be saturated at the dose levels used in thisstudy since neither plasma nor urinary formatelevels increased significantly with increasing dose.

The high levels of formic acid in urine result ina change in pH and are accompanied by changesin ammonia, and possibly calcium, excretion. Sim-ilar observations have been made previously inboth laboratory animals and in humans exposedto formic acid. Exposure has also been linked to


kidney damage although the exact mechanism bywhich formic acid damages the kidney is uncer-tain (Jacques, 1982; Liesivuori, 1986; Liesivuoriand Savolainen, 1987, 1991; Liesivuori et al.,1987, 1992). It has been shown that formic acidinhibits cytochrome c oxidase, the terminal oxi-dase of mitochondrial respiration (Nicholls, 1976;Erecinska and Wilson, 1980). The inhibition con-stant Ki is reported to be 1 mM, which is signifi-cantly lower than the urinary concentration of 20mM found in this study. As a result of inhibitingthis enzyme, ATP production is affected which inturn affects the levels of cAMP and the uptake ofCa2+ from the lumen. Ammonia is similarly af-fected, thus accounting for the increased excretionof these two ions in urine. Sustained inhibition ofthis enzyme is also believed to lead to histotoxichypoxia and ultimately structural kidney damage(Zitting et al., 1982). Formic acid is also reportedto affect the transport of a number of ions be-tween the lumen and proximal tubular cells (Kar-niski and Aronson, 1985; Schild et al., 1986,1987). Which of these mechanisms is correct re-mains to be established, but a link between expo-sure to formic acid and kidney damage has beenestablished in both animals and humans.

Renal toxicity is not normally reported follow-ing exposure to chemicals such as methanol andformaldehyde which are metabolised to formicacid (Liesivuori and Savolainen, 1991). However,the clearance of formic acid produced metaboli-cally from these chemicals is rapid and markedlydifferent from the high and sustained formic acidexposure which is seen in trichloroethylene treatedrats. It is not too surprising therefore that renaltoxicity is not associated with these otherchemicals.

It is interesting that the only effects detectablein the 28-day study were those related to formicacid excretion. Changes in urinary enzymes andthe other parameters which are known to besensitive to cysteine conjugates activated by b-lyase were unchanged at these cancer bioassaydose levels. It is possible therefore that formicacid excretion may provide an alternative plausi-ble mechanism for the chronic nephrotoxicity oftrichloroethylene in rats, and, since it is generallybelieved that the renal tumours are a direct result

of kidney damage, for the renal carcinogenicity ofthis chemical in the rat. Further studies are cur-rently underway to establish the source of theformic acid, to look in more detail at its effects onthe kidney, and to consider other species includ-ing humans.

Acknowledgements

These studies were sponsored by members ofthe European Chlorinated Solvent Association(Brussels, Belgium), the Halogenated Solvents In-dustry Alliance (Washington, DC) and the JapanAssociation for Hygiene of Chlorinated Solvents(Tokyo, Japan).

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