Hexachlorobenzene metabolism—Mainly in the rat

Fd Co.~met. T , xicol. VoI: 16. pp. 287-292. Pergamon Press 1978. Printed in Great Britain

Information Section ARTICLES O F G E N E R A L INTEREST HEXACHLOROBENZENE METABOLISM--MAINLY IN THE RAT • Hexachlorobenzene (HCB) is used as a fungicide, and is also a by-product of the industrial synthesis of many chlorinated hydrocarbons (Cited in F.C.T. 1976, 14, 351). It has hepatotoxic potential (ibid 1977, 15, 80) and studies of its metabolism are particularly important because of the suspicion that a metabolite may be responsible for the porphyrogenesis which is a feature of HCB intoxication (ibid 1976, 14, 352). Iatropoulos et al. (Envir. Res. 1975, 10, 384), in the course of a study of the absorption, transport and distribution of dichlorobiphenyl, dieldrin and HCB, gave a single intragastric dose of 150#g 14C-labelled HCB to male and female rats, which were examined 1-48 hours later. Little HCB was absorbed by the gastric and duodenal walls during the first hour after dosing, but after 3 hours increasing concentrations appeared in the cells lining the jejunum and ileum. Some t4C activity appeared in the liver and kidney, principally some 5 hours after dosing, but the highest levels were in the lymph nodes and adipose tissue and these levels were maintained throughout the observation period. Thus, absorption of H C B appears to be slow, with only minor involvement of the portal venous system; most absorption is effected by the lymphatic system and leads to deposition in body fat. Deposition in body fat was also a marked feature of the tissue distribution of HCB fed to rats for up to 15 weeks in daily doses of 0.5--32 mg/kg (KuiperGoodman et al. (Toxic appl. Pharmac. 1977, 40, 529). In the rats killed between weeks 3 and 15 of the feeding period, the tissue levels were relatively constant, those in adipose tissue, the liver, and the brain, kidney and spleen,, respectively, being some 200-500, 10-20 and 5-10 times higher than the serum levels. Tissue concentrations of HCB declined slowly during a 33-week period in which some of these rats were given an HCBfree diet. No striking difference between the sexes was apparent in the tissue-level determinations after a single HCB dose (Iatropoulos et al. Ioc. cit.) but in the feeding study HCB concentrations in the adipose tissue and brain were considerably higher in females than in males, and serum levels were ais6 generally higher in females. This paper (Kuiper-Goodman et al. Ioc. cit.) thus provides support for previous demonstrations of a sex-difference in the tissue accumulation of HCB in rats and in the greater sensitivity of females of this species to the toxic effects of this compound (Cited in F.C.T. 1976, 14, 351). Conflicting evidence on this aspect was presented, however, by Villeneuve & Newsome (Bull• eno. contam. & Toxicol. (U.S.) 1975, 14, 297), who gave pure HCB in daily oral doses of 500 mg/kg to groups of eleven rats and seven guinea-pigs of each sex for up to 16 days. On the basis of death rates and bodyweight loss, the male rats appeared slightly more sensitive than .the females, with eight males and five

females dying during treatment. There were no significant differences between the sexes, however, in the brain and liver levels of HCB, either in the animals that died or in the survivors, which were killed 24 hours after the last HCB dose. It seems likely" that the greater susceptibility of female rats compared with males is limited to the porphyrogenic properties of HCB. Porphyria only develops after about 3 weeks or more of HCB administration, depending on the dose, and would therefore not affect the results of acute or short-term studies like that of Villeneuve & Newsome (Ioc. cir.). These authors also reported that among the guinea-pigs, all of which died during treatment, the males lost more weight and accumulated more HCB in the brain and liver than the females. In comparison with the rat, tissue accumulation of HCB was lower in the guinea-pig, although the rat was less susceptible to the toxic effects of HCB in terms of deaths and body-weight loss. When female rats were fed pure HCB at a dietary level of 80 ppm from 2 weeks before mating until the killing of their second litter, examination of the pups from each litter at the age of 18 days showed the highest concentrations of HCB in the liver, followed in diminishing order by the kidney, lung, brain, spleen and heart (Mendoza et al. Enoir. Physiol. Biochem. 1975, 5, 460). No sex-determined differences in concentration were apparent. Liver concentrations of HCB were about 60ppm. The porphyrin concen.tration of the livers of exposed pups was about 2.5 times the control level, and was similar in males and females. The liver weight of offspring was raised significantly by HCB treatment of the dams, whereas the weights of kidney, brain, spleen and heart were reduced, the decrease in brain weight being greater in females than in males. With the exception of the work of Iatropoulos et al. (Ioc. cit.), which involved carbon-14 determinations after administration of [14C]HCB, all the studies mentioned above were based on the gas-chromatographic determination .of unchanged HCB in the blood or tissues. Several subsequent papers have been concerned with the identification of possible HCB metabolites in the tissues and excreta of rats. Engst et al. (Bull. eno. contain. & Toxicol. (U.S.) 1976, 16, 248) intubated male rats daily, with 8 mg HCB/kg for 19 days, after which the liver; kidneys, adrenals, heart, spleen and intestinal fat were isolated and n-hexane extracts were subjected to gas chromatography. Urine and faeces were collected for analysis during the secorid and third weeks of HCB treatment. Pentachlorobenzene and pentachlorophenol were found in low concentrations in the organs and tissues examined. The mean concentrations of HCB in fatty tissue and muscle were 82 and 17 ppm, respectively, while the total amounts of HCB determined in the major organs averaged 125, 21, 9, 1"5 and 0"5/~g in

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Articles of general interest

the liver, single kidney, spleen, heart, and single adrenal gland, respectively. Urine contained HCB and pentachlorophenol, which constituted the main metabolite, together with 2,3,4,6- and/or 2,3,5,6-tetrachlorophenol, 2,4,6-trichlorophenol, pentachlorobenzene and traces of 2,3,4- and other trichlorophenols. Small amounts of the glucuronides of 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol were .also present. Faeces contained relatively large amounts of HCB and a little pentachlorobenzene. The main degradation route for HCB in rats thus appears to involve conversion to pentachlorophenol and subsequently to tetra- and then trichlorophenols. Another metabolite isolated from the urine of male and female rats given an oral dose of 300 mg HCB/kg was conclusively identified as 2,4,5-trichlorophenol by Renner & Schuster (Toxic. appl. Pharmac. 1977, 39, 355), who thus confirmed and extended an earlier indication that traces of this compound were present in the urine of HCB-treated rats (Mehendale et al. J. agric. Fd Chem. 1975, 23, 261). In a more quantitative study of HCB metabolites, Koss et al. (Arch. Tox. 1976, 35, 107) gave female rats ip injections of ~4C-labelled HCB on 2 or 3 occasions over a period of 5 or 10 days, to provide a total dose of 260 or 390 mg/kg. Urine and faeces were collected for 4 weeks after the initial injection, and tissues were isolated for analysis at the end of this period. The mean excretion of labelled carbon totalled 7~o in the urine and 27~ in the faeces. In the latter, about 30~o of the activity was in the form

of metabolites and 70~ as unchanged HCB, while over 90~ of the urinary activity was in the form of metabolites, principally pentachlorophenol, tetrachlorohydroquinone and pentachlorothiophenol. In the tissues, the only metabolite detected in measurable amounts was pentachlorophenol (accounting for 10~o of the activity in blood, 3-5~/o in liver, 2 ~ in kidney, 1~ in brain and less than 0"l~d in body fat), most of the remaining activity being accounted for by unchanged HCB. At the end of the experimental period (4 weeks after the first dose), about 65~o of the administered dose remained, largely unchanged, in the body, about 18~o had been excreted unchanged in the faeces and some 15~o had been recovered as urinary and faecal metabolites. We are left again with the problem of relating these findings to the human situation. HCB has been shown to be a virtually universal residue in human body fat and a study carried out among children in various parts of Upper Bavaria (Richter & Schmid, ibid 1976, 35, 141) revealed concentrations of HCB in the blood ranging from 2"6 to 77.9 ppb (b = 109). Mean levels showed a rapid rise during the first 3 years of life but remained roughly constant .for children between the ages of 5 and 18 years, at 22 ppb for boys and 17 ppb for girls. These levels are of roughly the same order as those found in earlier studies of human blood samples. [P. Cooper--BIBRA]

A BREATH O F CADMIUM Occupational exposure to dusts containing cadmium (Cd) can result in a deterioration of lung function, as shown by the standard tests for forced vital capacity, forced expiratory volume at 1 second, and peak expiratory flow rate (Cited in F.C.T. 1977, 15, 479). Some further light has now been shed upon the mechanism by which Cd damages the lungs. Palmer et al. (Am. Rev. resp. Dis. 1975, 112, 173) exposed rats for 2 hours to an aerosol containing 0"1~ cadmium chloride (CdCI2) in physiological saline (generated from a 0.005 M solution and yielding an atmospheric concentration of 10mg CdClffm 3) and then killed batches of the animals for pulmonary investigation at intervals ranging from 1 hour to 10 days after treatment. The wet w~ght of the lungs of exposed animals (expressed as a percentage of terminal body weight) was doubled by day 4, but the only indication that this was due to inflammatory oedema was a transient increase in the fluid content of the lungs 24 hours after exposure. By day 10 the wet weight had dropped again almost to that found in untreated rats or in controls exposed to a saline aerosol, but by that time the dry weight of the Cdexposed lungs showed a significant increase. In rats given an ip injection of tritiated thymidine 30 minutes before they were killed, there was a marked appearance of the SH label in type II alveolar cells 24 hours after inhalation of CdClz; this reached a peak at day

3, declining to the basal control level by day 7. This indication of appreciable cellular proliferation after Cd exposure was supported by a marked increase in the lung content of DNA, which reached a peak on day 4. From day 2 onwards, 3H-labelled nuclei were present in the interstitial lung cells and in cells lying free in the alveoli as well as in the type II alveolar cells. Thus exposure to Cd aerosols evidently induced proliferation of lung cells, a development that may be related to fibrogenesis. In another experiment using the same concentration of CdCI2 aerosol (Hayes et al. ibid 1976, 113, 121), the lungs from exposed rats were homogenized for the determination of total extractable lipid and of malate, lactate, isocitrate and glucose-6-phosphate dehydrogenases. By day 4 after exposure, at a time when wet weight and total DNA content of the lungs had approximately doubled, total lipid, lactate dehydrogenase and glucose 6-phosphate dehydrogenase had also doubled. A peak in malate dehydrogenase concentration 1 hour after Cd exposure was followed by an equally sharp fall and a subsequent slow rise over the next 4 days. Enzyme activities had returned to control levels by day 10, but the content of extractable lipid remained elevated. The changes found were generally consistent with a non-specific lung injury involving the proliferation of type II cells, but the sharp initial rise in malate-dehydrogenase activity