Methanol and formic acid toxicity are characterized by metabolic acidosis (McMartin et al. I977; I980). Aci- dosis is mainly caused by the anaerobic metabolism ...
Br. |. exp. Path. (I987) 68, 853-86I
Kinetics and toxic effects of repeated intravenous
dosage of formic acid in rabbits J. Liesivuori, V.-M. Kosma*, A. Naukkarinen* and H. Savolainent Kuopio Regional Institute of Occupational Health, P.O. Box 93, SF-70701 Kuopio, Finland, *Department of Pathology, University of Kuopio, P.O. Box 6, SF-702 Kuopio, Finland and tInstitute of Occupational Health Sciences, University of Lausanne, CH-i005 Lausanne, Switzerland Ii
Received for publication I 8 September I986 Accepted for publication I2 June I987
Summary. Adult male rabbits were injected i.v. with I00 mg buffered formic acid per kg body weight daily for 5 days with 24 h between the doses. The fifth dose was labelled with '4C-formic acid. Rabbits were killed I, 2 and 20 h after the last injection. The highest formic acid concentrations were found one hour after the fifth dose. Total formic acid concentrations were always higher than radiometrically measured. The maximum concentrations of formic acid in brain, heart, kidney and liver were roughly similar to the concentration which inhibits half of the cytochrome oxidase activity in vitro. Histological studies clearly demonstrated the histotoxic changes at cellular level. Calcium deposits were detected in all organs of the injected rabbits. They were absent in control animals. It seems that the formic acid metabolism is slow and that it may cause sufficient hypoxic acidosis to allow the calcium influx and cellular damage.
Keywords: formic acid, hypoxic acidosis, intracellular calcium Formic acid has an increasing significance in the body (Martin-Amat et al. 1978; Eells et al. many chemical processes as a raw material I 9 8 I; Liesivuori I 98 6; Liesivuori & Savolaiand it is used also as such, e.g. in the control nen I986). The accumulation of formate of acidity in resin production and in the varies in different species depending on the agriculture (Chalkins I984; Liesivuori & effectivety of methylotropic metabolism through the folic acid cycle (Clay et al. I 9 75; Kettunen I983). As a metabolic end-product of methanol Billings & Tephly 1 9 79; Quayale I 980; Eells formic acid seems to account for the metha- et al. I98I). nol toxicity (Martin-Amat et al. I978; BillFormic acid is an inhibitor of the cytoings & Tephly I979). Methanol and formic chrome oxidase complex (Erecifiska & Wilacid toxicity are characterized by metabolic son I980). The inhibitory effect leads to the acidosis (McMartin et al. I977; I980). Aci- so-called histotoxic hypoxia. This may espedosis is mainly caused by the anaerobic cially affect organs with high oxygen conmetabolism rather than by protons from sumption, e.g. kidney, brain and heart formate (Savolainen I982). Metabolically (Savolainen & Zitting I980; Zitting & Savogenerated formic acid is cleared slowly from lainen I980; Zitting et al. I982). Correspondence: Jyrki Liesivuori, Kuopio Regional Institute of Occupational Health, P.O. Box 93, SF-7070I Kuopio, Finland.
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A very significant concentration gradient of calcium ions across the cell membrane is maintained under physiological conditions (Rasmussen I983). Increased cytosolic calcium concentrations cause various metabolic and physiological responses because of the ion's regulatory roles (Qi et a]. I983; Silinsky I985; Simonson et al. I985). Hypoxia affects profoundly the ion distribution in brain (Hansen I985). Especially, the influx of calcium may be an early effect of ischaemia (Farber et al. I 98 I; Dienel I 984). Calcium accumulation in ischaemia has been observed also in renal cortex (Schieppati et al. I985; Jones I986) and in cardiac myocytes (Cheung et al. I986a). The mechanism of the increased permeability of cell membrane to calcium ions are not exactly known although increased H+ concentration is a contributing factor (Iijima et al. I986). In this work, we studied the effects and accumulation of formic acid in rabbits after five daily dosages of formic acid.
Materials and methods Fifteen male New Zealand rabbits (3070± 220 g,±s.d.) were given i ml of freshly diluted solution containing i oo mg formic acid/ kg body weight by injection into ear vein on 5 consecutive days with 24 h between the doses. Formic acid (puriss. p.a., Fluka AG, Buchs, Switzerland) was adjusted to pH 7.4 by O.OI M phosphate buffer. The fifth dose was labelled with '4C-formate (specific activity 58 mCi/mmol, 3.1 uCi/kg, Amersham International, Buckinghamshire, UK). Three control rabbits were injected with buffer solution. The animals received food and water ad libitum during the experiment. The animals were killed under barbiturate anaesthesia I, 2 and 20 h after the fifth dose. Blood samples were drawn into heparinized tubes. Urine specimens were taken from the bladder. Brain, heart, kidney, and liver were taken at autopsy and weighted. Samples (i g) of each organ were homogenized in distilled water by Ultra-Turrax
(Janke & Kunkel GmbH, Staufen, FRG) and centrifuged. An aliquot of i ml was taken for the measurement of radioactivity with a LKB Liquid Scintillation Counter (Wallac, Turku, Finland). Another aliquot (I ml) of the samples was stored at -2 5°C for formic acid analysis. The concentrations of formic acid were determined after the methylation of the acid and its conversion to N,N-dimethylformamide using a Hewlett-Packard 588o capillary gas chromatograph (Liesivuori i986). The method for formic acid assay had recoveries of 87-95% depending on the organ. The variation coefficient was 0.03-0.05. The results were corrected for the recoveries. Biopsies were taken from the main lobe of the liver, from the cortex of the kidney, from the myocardium and from the brain. The samples were fixed in 4% buffered formalin, and routinely processed for light microscopy. Sections 5 gum thick were stained with haematoxylin-eosin and by von Kossa's method for calcium. The samples were examined with a Leitz Dialux 22 microscope equipped with a Leitz Vario-Orthomat photographic system. For electron microscopy, the samples were fixed by immersion in 2% phosphate-buffered glutaraldehyde, post-fixed in I% osmium tetroxide, dehydrated, and embedded in Epon. The ultrathin sections were stained with uranyl acetate and lead citrate. The specimens were studied with a Jeol JEM iooS electron microscope. Results Except in brain, peak concentrations of formic acid were measured one hour after the fifth dosage (Table i). Urine samples showed the highest concentrations at all times. In brain, the highest concentration of formic acid was detected 2 h after the dosing. The decay of blood formic acid between 2 h and 20 h was more rapid than in other organs. The radiometrically detected formic acid concentrations were always lower than chemically found acid. Daily residual fractions
Formic acid dosaqe in rabbits
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Table i. Formic acid concentrations in tissue compartments after five daily intravenous doses Time after the
fifth dose (h)
Total (pmol/g)
14C-labelled (pmol/g) Difference
I
0.7±0.4
0.3±0.I
2 20
0.5+0.2
0.2+0.I
0.2±0.1
0.I ±0.05
I
2
I.1±0.4 I .3 ± o.6
0.4±0.2 o.6 ± 0.3
20
0.7±0.4
0.1±0.05
0.4±0.2 0.5 ±0.2 0.4±0.I
Blood
Brain
0.4±0.I 0.2+0.I 0.2±0.05
I
o.8±0.3
o.6±0.3
0.6±0.2
2 20
0.7±0.3
0.4±0.2
0.5±0.2
0.1±0.05
0.3±0.I 0.3±0.I
I.5+0.5 I.I ±0.4 0.3±0.05
0.9±0.5 0.7±0.4
0.4±0.2 0.5 ±0.2
0.2±0.05
0.1±0.05
I
I.7±0.7
o.8±o.5
o.6±0.4
2 20
i.o±o.6 0.4±0.I
0.7±0.2 0.2±0.05
o.8±0.4 0.2±0.05
I
44±22
27±I2
I7±8
2 20
32±I2
25± 8 0.1± .OI
0.5±0.I
Heart
Liver
I 2
20
Kidney Urine
0.6±0.2
9±6
Rabbits were injected i.v. with IOO mg/kg buffered formic acid daily for 5 d with 24 h between the doses. Samples were taken I, 2 and 20 h after the fifth dose of I4C-labelled formic acid. Each figure is the mean of five rabbits ± s.d. The difference calculated in each animal between the chemically determined acid and the radiometrical result shows the accumulation of formate from the four previous injections. Formic acid concentrations in the control rabbits were under the detection limit
(0.05 mmol/l). compared favourably with the remaining labelled formic acid on the fifth day (Table i). These facts may point to an accumulation of formic acid from one injection to another and to a slower metabolism of the acid in the brain than in the circulation. Kidneys actively secreted formic acid in the urine. The partition ratio (formic acid concentration in tissue devided by concentration in blood) h after the fifth dose was for kidney 2.4, for liver 2.1, for brain I.6 and for heart I. I. The ratios indicate the large variation in the distribution of the acid in body fluids. Aggregates of haematoxylin positive calcium were frequently observed in the lumen i
of both distal and proximal tubules in the microscopic examination of kidneys. The epithelium of distal tubules showed atrophy and flattening, especially in the vicinity of the calcium aggregates (Fig. i). In liver, calcium surrounded by inflammatory cells was found mainly in the portal tract and around the central veins. Calcium casts appeared also in the walls of arteries and bile ducts (Fig. 2). Calcium deposits were observed in the endocardium, and they were surrounded by inflammatory cells (Fig. 3). In the samples taken from the frontal pool and temporal lobe cortex of the cerebral hemispheres as well as from the hippocampus and
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Fig. i. a,Distal tubules from a kidney of a control rabbit; b, Calcium in the lumen of distal tubules in the formic acid exposed animal. The tubular epithelium is atrophied (arrow). H & E, x 750.
posterior cerebellar vermis calcium deposits were also seen, although other histological changes were not as clear as seen in the other organs. In the electron microscopic observations, increased numbers of myelin figures were seen in the cardiac cells of the exposed rabbits as an early effect of degeneration
(Fig. 4).
The hepatocytes and nephrocytes of the exposed animals showed only minor changes in electron microscopic analysis. Proliferation or vacuolization of smooth endoplasmic reticulum were not observed in the liver cells. The mitochondria and peroxisomes were normal and increased amount of
lysosomal material was not detected. No ultrastructural alterations could be identified in the brain samples. Discussion The rat is a poor model for the study of toxicity of the methanol or formic acid because it has a very effective methylotropic metabolism through the folic acid cycle (Quayle I980). Rabbits, dogs and monkeys are closer to man in this respect (Lund 1948a, b; Martin-Amat et al. I978; McMartin et al. i980; Liesivuori I986; Liesivuori & Savolainen I986; I987). The species difference in formate accumulation cannot be
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Fig. 2. a, Central vein in the liver of a control rabbit; b, Extracellular calcium is surrounded by inflammatory cells in the liver of the formic acid exposed animal. H & E, x 750.
explained by different rates of metabolism (Clay et al. I975). However, the maximal observable rate of formate oxidation to carbon dioxide in the rat is at least twice the rate seen in the monkey. The difference in the metabolic capacity may be due to a relative folate deficiency which exists, e.g. in the monkey (McMartin et al. I977; Eells et al. I98I). Rabbits try to offset the hypoxic acidosis by deepening respiration and increased exhalation of carbon dioxide so that the clinical toxicity of formic acid doses can be easily determined by observing the respiratory efforts of the animals. The single formic acid dose (2.2 mmol/kg) in our study was rather small compared with
those infused (3.I mmol/kg/h) in the monkeys by Martin-Amat et al. (I978). They produced formate concentrations in the blood ranging between io mmol/l and 30 mmol/i after I0 h, whereas the concentrations were from 0.2 to 0.7 mmol/l in our study (Table i). Formic acid is also oxidized to carbon dioxide faster in rabbits than in monkeys (Lund 1948a; Martin-Amat et al. I978). In earlier studies formic acid was only assayed in body fluids, probably because of
analytical difficulties (Makar et al. I975; Abolin et al. I980). No data has been found for comparison of the formate concentrations determined by us for different tissues.
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Fig. 3. a, Heart muscle of a control rabbit; b, Calcium aggregates are surrounded by inflammatory cells in the endocardium of the formic acid exposed animal. H & E, x 750.
However, our results have been obtained by two independent methods, i.e. by radiometric and chemical analyses. Formic acid is dissociated in water phase (PKa 3.67) which could explain the low concentrations of the acid in myocardium (Table i). The blood brain barrier seems to prevent formic acid from distributing rapidly into the brain (Table I). The Ki for the inhibition of the mitochondrial cytochrome oxidase activity by formic acid is i mmol (Erecin'ska & Wilson I980). The maximum concentrations of formic acid in brain, heart, liver and kidney are roughly similar to that (Table i). This could very well cause impaired oxidative metabolism in its turn causing increased anaerobic glycolysis with
associated acidosis (Hochachka & Mommsen I983).
Calcium overload has been implicated in ischaemic injury and cell death. An increase in free cytosolic calcium will activate calcium-dependent phospholipases, resulting in the breakdown of cell membranes and the accumulation of free fatty acids and lysophospholipids that are toxic to the cell (Cheung et al. i 986b). In addition, the secretion of acetylcholine quanta (Silinsky I985), the protein kinase reaction (Qi et al. I983) and the activation of so-called calcium-activated neutral proteinase (Simonson et al. I985) are regulated by calcium. It has been suggested that mitochondrial dysfunction may be secondary to calcium over-
Formic acid dosage in rabbits
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Fig. 4. a, Electron micrograph from a control rabbit showing heart muscle. x 8ooo. b, Myelin figure (arrow) between cardiac muscle filaments in the formic acid exposed animal. x i 6 000.
load in the mitochondria (Cheung et a]. I 98 6a, b). The increased H+ concentration is a contributing factor although the mechanisms of the increased permeability of cell membrane to calcium ions are not exactly known (Iijima et al. I986). This notion is
supported by the intracerebral calcifications associated with renal tubular acidosis by congenital carboanhydrase II deficiency (Sly et al. I986). A very slight swelling of mitochondria could be seen only in the myocardium (Fig.
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4). The kidney effects differ from those in liver possibly because of the much higher oxygen consumption in the kidney. Hypoxic changes in kidney include swelling and degeneration in the epithelial cells of the tubuli (Zitting et al. I 982) which was also seen in our experiment. Myelin figures observed in myocardium (Fig. 4) are the early signs of degeneration caused by hypoxia (Levene et at. I 98 5). In conclusion, formate toxicity may be a suitable model for histotoxic hypoxia. Its dose can be readily regulated, it can be analysed in biological samples, and the results can have a direct bearing in the exposed workers with known internal formate burdens (Liesivuori & Savolainen I987).
Acknowledgements We thank Ms Eeva Ruotsalainen, Ms Sirkka Roivainen, Ms Terttu Viitanen and Mr Heikki Pekonen for their skilful technical assistance and the Finnish Work Environment Fund for financial support. References ABOLIN C., McRAE J.D., TOZER T.N. & TAKKI S. (I980) Gas chromatographic head-space assay of formic acid as methyl formate in biologic fluids: Potential application to methanol poisoning. Biochem. Med. 23, 2I9-222. BILLINGS R.E. & TEPHLY T.R. (I979) Studies on methanol toxicity and formate metabolism in isolated hepatocytes. The role of methionine in folate-dependent reactions. Biochem. Pharmacol. 28, 2985-2991. CHALKINS W.H. (i 984) Chemicals from methanol. Catal. Rev. Sci. Eng. 26, 347-358. CHEUNG J.Y., LEAF A. & BONVENTRE J.V. (I986a)
Mitochondrial function and intracellular calcium in anoxic cardiac myocytes. Am. J. Physiol. 250, CI8-C25. CHEUNG J.Y., BOVENTRE J.V., MALIS C.D. & LEAF A. (I986b) Calcium and ischemic injury. N. Engl. J. Med. 314, I670-I676. CLAY K.L., MuRPHY R.C. & WATKINS A.D. (I975) Experimental methanol toxicity in the primates: Analysis of metabolic acidosis. Toxicol. Appi. Pharmacol. 34, 49-6 i. DIENEL G.A. (I984) Regional accumulation of
calcium in postischemic rat brain. J. Neurochem. 43,9I3-925. EELLS J.T., MAKAR A.B., NOKER P.E. & TEPHLY T.R. (I98I) Methanol poisoning and formate oxidation in nitrous oxide-treated rats. J. Pharmacol. Exp. Ther. 217, 57-6I. ERECIN4SKA M. & WILSON D.F. (I980) Inhibitors of cytochrome c oxidase. Pharmacol. Ther. 8, I-20.
FARBER J.L., CHIEN K.R. & MITTNACHT S. (I98I) The pathogenesis of irreversible cell injury in ischemia. Am. J. Pathol. 102, 27I-28I. HANSEN A.J. (I985) Effect of anoxia on ion distribution in the brain. Physiol. Rev. 65, IOI-I48. HOCHACHKA P.W. & MOMMSEN T.P. (I983) Protons and anaerobiosis. Science 219, I39II397. IIJIMA T., CIANI S. & HAGIWARA S. (I986) Effects of external pH on Ca channels. Experimental studies and theoretical considerations using a two-site, two-ion model. Proc. Nati. Acad. Sci. USA. 83, 654-658. JONES D.P. (I986) Renal metabolism during normoxia, hypoxia, and ischemic injury. Ann. Rev. Physiol. 48, 33-50. LEVENE C.I., BARTLET C.P., FORNIERI C. & HEALE G. (I985) Effect of hypoxia and carbon monoxide on collagen syntesis in cultured porcine and bovine aortic endothelium. Br. J. exp. Path. 66,
399-408.
LIESIVUORI J. (I986) Slow urinary elimination of
formic acid in occupationally exposed farmers. Ann Occup. Hyg. 30, 329-333. LIESIVUORI J. & KErrUNEN A. (I983) Farmers' exposure to formic acid in silage making. Ann. Occup. Hyg. 27, 327-329. LIESIVUORI J. & SAVOLAINEN H. (I986) Urinary excretion offormic acid in rabbits. Acta Pharmacol. Toxicol. 58, I6I-I62. LIESIVUORI J. & SAVOLAINEN H. (I987) Urinary formic acid as an indicator of occupational exposure to formic acid and methanol. Am. Ind. Hyg. Assoc. J. 48, 32-34. LuND A. (I 948a) Metabolism of methanol and formic acid in rabbits. Acta Pharmacol. Toxicol. 4, 99-107. LuND A. (1948b) Metabolism of methanol and formic acid in dogs. Acta Pharmacol. Toxicol. 4, I08-I2I. MAKAR A.B., MCMARTIN K.E., PALESE M. & TEPHLY T.R. (I975) Formate assay in body fluids: Application in methanol poisoning. Biochem. Med. 13, II7-126. MARTIN-AMAT G., MCMARTIN K.E., HAYREH S.S., HAYREH M.S. & TEPHLY T.R. (1978) Methanol
Formic acid dosage in rabbits poisoning: Ocular toxicity produced by formater. Toxicol. Appl. Pharmacol. 45, 20I-208. McMARTIN K.E., MARTIN-AMAT G., MAKAR A.B. & TEPHLY T.R. (I977) Methanol poisoning. V. Role of formate metabolism in the monkey. J.
Pharmacol. Exp. Ther. 201, 564-572. McMARTIN K.E., AMBRE J.J. & TEPHLY T.R. (I980) Methanol poisoning in human subjects. Role of formic acid accumulation in the metabolic acidosis. Am. J. Med. 68, 4I4-4I8. Qi D-F., SCHATZMAN R.C., MAZZEI G.J., TURNER R.S., RAYNOR R.L. LIAO S. & Kuo J.F. (I983) Polyamines inhibit phospholipid-sensitive Cadependent protein kinases. Biochem. 1. 213, 28I-288. QUAYLE J.R. (I980) Aspects of the regulation of methylotrophic metabolism. FEBS Lett. II17, Ki6-K27. RASMUSSEN H. (I983) Cellular calcium metab-
olism. Ann. Intern. Med. 98, 809-8I6. SAVOLAINEN H. (I982) Lactate and formate in methanol poisoning. Lancet i, 562. SAVOLAINEN H. & ZITrING A. (I980) Glial cell effects of subacute formic acid vapour exposure.
Acta Pharmacol. Toxicol. 47, 239-240. SCHIEPPATI A., WILSON P.D., BURKE T.J. & SCHRIER R.W. (i 98 5) Effect of renal ischemia on cortical
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microsomal calcium accumulation. Am. J. Physiol. 249, C467-C483. SILINSKY E.M. (I985) The biophysical pharmacology of calcium-dependent acetylcholine secretion. Pharmacol. Rev. 37, 8I-I32. SIMONSON L., BAUDRY M., SIMAN R. & LYNCH G. (I985) Regional distribution of soluble calcium activated proteinase activity in neonatal and adult rat brain. Brain Res. 327, I53-159. SLY W.S., WHYTE M.P., SUNDARAM V., TASHIAN R.E., HEWETT-EMMETT D., GUIPAUD P., VAINSEL M., BALUARTE H.J., GRUSKIN A., AL-MOSAWI M., SAKATI N. & OHLSSON A. (I986) Carbonic anhydrase II deficiency in i 2 families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. N. Engi. J. Med. 31I3, I39-I45. ZITTING A. & SAVOLAINEN H. (I980) Biochemical effects of subacute formic acid vapor exposure. Res. Commun. chem. Pathol. Pharmacol. 27,
I57-I62. ZITTING A., SAVOLAINEN H. & NICKELS J. (I982) Biochemical and toxicological effects of single and repeated exposures to polyacetal thermodegratation products. Environ. Res. 29, 28 7-296.
