434 Articles of general interest--fd Cosnrer. Toxicol. Vol. 18, no. 4

decrease surface tension and to stabilize surface forces collapse. Collapse of the alveoli may, in turn, cause between large and small alveoli during the inflation/ respiratory distress and morphological changes, the deflation cycle (Morgan, New’Eng. J. Med. 1971, 284, latter (‘pseudofibrosis’) resulting directly from the 1185). Using female rats and the exposure procedure apposition of collapsed alveolar walls. True fibrosis. described above (except that exposure was terminated however, is a later event, and certain early changes after 45 days), Le Mesurier er al. (lot. cit.) found that are not accounted for by this hypothesis either. Thus by day 5 there was a marked reduction in the yield of both the vascular endothelium and interstitial fibro- surfactant obtained by endobronchial lavage. There- blasts showed early degeneration but apparently re- after the yield continued to decline, reaching a mini- covered the fibroblasts being stimulated later to an mum by day 15. Subsequently there was some tluc- excessive level of collagen production. The tentative tuating recovery, and between days 35 and 45 a rela- suggestion is made, therefore, that different factors in tively constant yield, approximately 50% of the con- the petrol vapour may be acting independently on trol mean, was maintained. Chromatography of the these different cells of the pulmonary parenchyma. surfactant showed no qualitative change in its phos- Further studies on this topic will be of interest, not pholipid composition during the course of the ex- merely in connection with assessing the toxic hazard posure. of petrol inhalation but as a possible contribution to

While the mechanisms underlying these effects can- a greater understanding of the syndrome of fibrosing not be said to be established, these workers deduced alveolitis in man. Such studies may perhaps provide several hypotheses from their findings. One possible confirmation of the evidence presented here, which rationale for the series of events observed is that sur- seems to point to the type 2 pneumocyte as the factant secretion may be inhibited by the direct effect primary target for some constituent of petrol vapour. of the petrol vapour on the surfactant-producing organelles of the type 2 pneumocytes and that the resulting deficiency of surfactant precipitates alveolar [P. Cooper-BIBRA]

STYRENE AND THE CHROMOSOME

The primary metabolite of styrene, its epoxide, has been found to be a potent mutagen in a number of test systems, with and without metabolic activation (Cited in F.C.T. 1978, 16, 300; ibid 1978, 16, 397; ibid 1979, 17, 298). Styrene itself has been found to in- crease the chromosoinal aberration rate of the lym- phocytes of occupationaily-exposed workers (ibid 1979. 17, 299), but the results of experimental studies have been equivocal although they have established that the presence of a metabolic activation system is an undoubted prerequisite of in aitro demonstrations of styrene mutagenicity.

Loprieno et al. (Stand. J. Work Enair. Hlth 1978, 4, Suppl. 2, 169) studied the incidence of chromosomal aberrations in the bone-marrow cells of mice given a single dose of styrene or styrene oxide by gavage and killed 24 hr after treatment. Styrene oxide caused a significant increase in the number of aberrations at doses of 50, 500 and lOOOmg/kg. A dose-dependent response was found although the highest dose was toxic. The aberration rate was unaffected by styrene. By contrast, Fabry et a/. (Mutation Res. 1978, 51, 377) gave adult male mice a single up injection of 250mg styrene oxide/kg and found no increase in the number of chromosomal aberrations in bone-marrow cells after 1, 2, 6 or 13 days but no results were shown either for positive or for negative controls]. Similarly, largely negative results were obtained by Norppa et al. (Chemico-Biol. Interactions 1979, 26, 305) using the bone-marrow of Chinese hamsters. The animals were exposed to 25, 50, 75 or 1OOppm styrene oxide for 9hr(6hrondayland3hronday2)or21hr(6hron days 1,2 and 3 and 3 hr on day 4). At the lowest dose the effects of exposure for 3 wk (6 hr/day. 5 days/wk) were also studied, and at the highest dose only the 9 hr duration was used because of serious signs of

poisoning in the animals. The animals were killed im- mediately after the last exposure. None of the animals treated by inhalation showed any significant increase in the frequency of cells with chromosomal aberra- tions. However in animals given a single ip injection of 500 mg styrene oxide/kg there was a significant in- crease in the number of chromosomal aberrations in the cells of animals that died 18-22 hr after treatment but not in those that were killed after 24 hr.

Chromosomal aberrations were also observed in human lymphocytes taken from a single donor and exposed to 0.1 or 0.5 mM-Styrene oxide for 48 hr (Fabry et al. lot. cit.). Most of the anomalies were chromatid gaps and breaks. Linnainmaa et al. (Muta- tion Res. 1978. 58, 277) studied human lymphocytes exposed to 2.6mh+styrene during the entire culture period or to 0.7 mM-styrene oxide during the last 8 hr of culture (both toxic levels). The styrene treatment induced chromosome breaks, and styrene oxide caused severe chromosome destruction resulting in pulverized chromosomes. In many metaphases that did not show chromosomal fragmentation the chromosomes acquired a non-specific banded stain- ing, and with both compounds there was a significant increase in the number of interphase cells with micro- nuclei or nuclear bridges. Onion (Allium cepa) root-tip cells were also used by Linnainmaa et al. (lot. cit.) to study the production of chromosomal aberrations in uiuo. The results with this sytem were, for both styrene and styrene oxide, very similar to those obtained with human lymphocytes in vitro.

These results and those of previous studies in rats and in man (Cited in F.C.T. 1979, 17, 299) show that both styrene and its oxide undoubtedly produce chro- mosomal aberrations under certain conditions. It seems likely that the assortment of results obtained

Articles of general interest--Fd Cosmer. Toxicol. Vol. 18. no. 4 435

arises from the widely different routes, levels and dur- ations of exposure used.

Styrene did not increase the mutation rate of Sal- monella ryphimurium strain TA1535 at concentrations ranging from I x 10e3 to IO~mol/plate either with or without S-9 mix (Loprieno er al. lot. cit.). Survival ranged from 0.8 to 100°? under these conditions. Sty- rene oxide was mutagenic both with and without the S-9 mix at levels of l-100 pmol/plate and the response was dose-related except at the highest dose-level which was extremely toxic. These results support those of Stoltz et al. (Bull. enuir. Contam. Toxicol. 1977. 17, 739; Cired in F.C.T. 1978, 16, 397) but con- tradict those of De Meester et al. (Mutation Res. 1977, 56, 147; Cited in F.C.T. 1979, 17, 298) who found that styrene itself was mutagenic towards this strain when a metabolic activating system was used.

In cultured Chinese hamster bone-marrow cells there was no increase in the mean number of sister- chromatid exchanges in any of the styrene oxide inha- lation exposure groups used by Norppa et al. (lot. cit.). However the group given 500 mg styrene oxide/kg by ip injection showed a marginally signifi- cant (P < 0.05) increase in the number of sister- chromatid exchanges, but the increase was only seen in one out of six animals. Styrene oxide was pre- viously shown to be a potent inducer of sister-

chromatid exchanges in oitro in Chinese hamster ovary cells (Cited in F.C.T. 1979, 17, 298).

Fabry et al. (lot. cit.) found no evidence of muta- genicity when polychromatic erythrocytes from 8-wk-old mice were examined 30 hr after a single ip injection of 250 mg-styrene oxide/kg. They also obtained negative results of the meiotic chromosomes of treated male mice and in dominant lethal tests per- formed 1-3 wk after the injection. DNA repair in cul- tured human (EUE) cells was stimulated by styrene oxide at a level of 4.4 mM, but styrene did not increase DNA repair even in the presence of a metabolic acti- vation system (Loprieno et al. lot. cit.). These authors also found that styrene oxide, but not styrene, pro- duced point mutations in the Chinese hamster cell line V-79. They consider that the lack of mutagenic activity of styrene itself may be due to rapid conver- sion of its active metabolite. styrene oxide, to phenyl- ethylene glycol. The latter conversion, which is acti- vated by microsomal epoxide hydratase, occurs at a faster rate than that of styrene to styrene oxide. More- over, it has been shown (Cited in F.C.T. 1978, 16, 300) that epoxide-hydratase activity is markedly increased by the treatment of rats with styrene but is largely unaffected by similar treatment with styrene oxide.

[P. Cooper-BIBRA]

THE VARYING FATE OF VINYLIDENE CHLORIDE

Vinylidene chloride (l,l-dichloroethylene; VDC) is used as a monomer in plastics manufacture, particu- larly for the production of wrapping films. Acute inhalation of VDC has been shown to decrease gluta- thione stores in the rat ,liver (Jaeger er al. Expl mol. Path. 1974, 20, 187) and inhalation of 25 ppm VDC for 52wk has been associated with the development of malignant tumours of the kidney in male mice, although 200 ppm had no such effect in rats (Maltoni et al. Medna Lau. 1977,&3,241). Several recent studies of VDC have highlighted the ways in which its metab- olism differs in mice and rats as well as the effects of inadequate food intake and of various routes of ad- ministration on its fate in rats. In other work, the species differences have been reflected in the results of short-term mutagenecity tests on VDC.

In the study by Jaeger er al. (lot. cit.). depletion of hepatic glutathione stores by VDC and the ac- companying hepatotoxicity were found to be exacer- bated by a period of fasting prior to VDC exposure. Later McKenna rt al. (Toxic. appl. Pharmac. 1978. 45, 599 & 821) studied the pharmacokinetics of VDC ad- ministered by inhalation or orally to both fed and fasted rats. In the inhalation study (p. 599). groups of male rats fed normally or fasted for 18 hr before treat- ment were exposed to 10 or 200 ppm [14C]VDC vapour for 6 hr. and the elimination of 14C activity was studied for the following 72 hr.

After exposure to IOppm, about 98”~; of the ab- sorbed VDC was converted to non-volatile metab- elites both in normal and fasted rats. However. after exposure to 200ppm. the proportion of VDC metabo-

lized was significantly lower and showed a significant difference between the fed and fasted rats, at 96 and 92% respectively of the total amount absorbed. There was also a significant difference between the total amounts absorbed by the fed and by the fasted rats, the total body burdens in the fasted rats being lower at both exposure levels. Despite the markedly higher pulmonary elimination of unchanged [‘4C]VDC by fasted rats exposed to 2OOppm, compared with rats exposed to 10 ppm, the former also showed a higher level of retention of 14C activity. Retention in the fed rats exposed to 200 ppm, however, differed little from that in the IO-ppm groups. The main sites of VDC retention in all the groups were the liver, kidneys and lungs. Liver and kidney lesions developed after 200-ppm exposure of the fasted but not the fed rats. No such effect resulted in any rats treated with IOppm VDC. The affected livers showed centrilobu- lar necrosis and the kidneys marked degeneration of the proximal tubular epithelium. The liver lesions were associated with a higher level of covalently bound 14C activity than was seen in fed rats and the levels in the latter were in turn considerably higher than those in normally fed rats exposed to IOppm VDC.

Similar results were obtained in rats given an oral dose of 1 or 50mg [14C]VDC/kg (McKenna er al. lot. cit. p. 82 I ). In normally fed rats the proportions of the lower and higher dose exhaled unchanged were 1-3 and 19”:,. respectively, and the metabolism of the larger dose was further reduced (29’4 being exhaled unchanged) in rats fasted for 18 hr before treatment.