Hazard identification by methods of animal-based toxicology.

This paper is one of several prepared under the project "Food Safety In Europe: Risk Assessment of Chemicals in Food and Diet" (FOSIE), a European Commission Concerted Action Programme, organised by the International Life Sciences Institute, Europe (ILSI). The aim of the FOSIE project is to review the current state of the science of risk assessment of chemicals in food and diet, by consideration of the four stages of risk assessment, that is, hazard identification, hazard characterisation, exposure assessment and risk characterisation. The contribution of animal-based methods in toxicology to hazard identification of chemicals in food and diet is discussed. The importance of first applying existing technical and chemical knowledge to the design of safety testing programs for food chemicals is emphasised. There is consideration of the presently available and commonly used toxicity testing approaches and methodologies, including acute and repeated dose toxicity, reproductive and developmental toxicity, neurotoxicity, genotoxicity, carcinogenicity, immunotoxicity and food allergy. They are considered from the perspective of whether they are appropriate for assessing food chemicals and whether they are adequate to detect currently known or anticipated hazards from food. Gaps in knowledge and future research needs are identified; research on these could lead to improvements in the methods of hazard identification for food chemicals. The potential impact of some emerging techniques and toxicological issues on hazard identification for food chemicals, such as new measurement techniques, the use of transgenic animals, assessment of hormone balance and the possibilities for conducting studies in which common human diseases have been modelled, is also considered.

Thunderstorm-related asthma--the epidemic of 24/25 June 1994.

BACKGROUND: A large epidemic of asthma occurred following a thunderstorm in southern and central England on 24/25 June 1994. A collaborative study group was formed. OBJECTIVES: To describe the epidemic and the meteorological, aerobiological and other environmental characteristics associated with it. METHODS: Collation of data from the Meteorological Office, the Pollen Research Unit, the Department of the Environment's Automatic Urban Network, from health surveillance by the Department of Health and the National Poisons Unit, from clinical experience in general practice and hospitals, and from an immunological study of some of the affected cases from north east London. RESULTS: The thunderstorm was a Mesoscale Convective System, an unusual and large form of storm with several centres and severe wind gusts. It occurred shortly after the peak grass pollen concentration in the London area. A sudden and extensive epidemic occurred within about an hour affecting possibly several thousand patients. Emergency services were stretched but the epidemic did not last long. Cases had high serum levels of IgE antibody to mixed grass pollen. CONCLUSION: This study supports the view that patients with specific IgE to grass pollen are at risk of thunderstorm-related asthma. The details of the causal pathway from storm to asthma attack are not clear. Case-control and time series studies are being carried out.

Chloracne caused by ingestion of olive oil contaminated with PCDDs and PCDFs.

1. All members of a Spanish family (father, mother and six children) developed chloracne. 2. The causative agent was found to be the family's stock of olive oil, which had become contaminated with polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), pentachlorophenol, and hexachlorobenzene. 3. The more highly chlorinated PCDDs, in particular octachlorodibenzo-p-dioxin, were the predominant congeners in the oil. 4. Three members of the family exhibited either an overt or a sub-clinical disturbance of kidney function. The father also had a chronic respiratory problem. These changes could not be unequivocally attributed to the PCDDs. 5. Experimental toxicity of the oil was limited to the development of an hepatic porphyria in mice. 6. A serum sample, taken 5 years after consumption of the oil ceased, contained high levels of the PCDDs and PCDFs. Extrapolation back to ingested dose was used to validate dosage estimates. 7. The use of toxicity equivalence factors (TEFs) provided estimates of cumulative dosage to produce chloracne as 0.13-0.31 micrograms 2378-TCDD kg-1 (using EPA TEFs) or 6.7-16 micrograms 2378-TCDD kg-1 (using Nordic/NATO TEFs). 8. This is the first incident in which human toxicity is related primarily to ingestion of PCDDs and for which estimates of dosage can be made.

Comparative acute nephrotoxicity of Penicillium aurantiogriseum in rats and hamsters.

Air-dried mycelium of Penicillium aurantiogriseum2, grown as a surface culture on yeast extract-sucrose medium, was incorporated into powdered diet and fed to rats and hamsters for different periods up to 28 days. At intervals, animals were anaesthetized and the kidneys fixed in situ by perfusion. In rats, the fungus produced scattered exfoliation of pyknotic cells and an increased frequency of mitotic figures involving the pars recta segment of proximal tubular epithelium. This lesion was detectable as early as three days after beginning of treatment and was well developed by 14 days. No degenerative tubular change or mitogenic effect was observed in hamsters, even after feeding for 35 days; and there was no apparent renal pelvic or interstitial lesion in either species.

Sex-linked hepatic uroporphyria and the induction of cytochromes P450IA in rats caused by hexachlorobenzene and polyhalogenated biphenyls.

A marked sex difference in the development of uroporphyria occurred after administration of polychlorinated and polybrominated biphenyls (PCBs and PBBs), as well as hexachlorobenzene (HCB), to F344 rats for 15 weeks. Thus the propensity of female rats to develop uroporphyria appears to be a general response to this class of halogenated chemicals. A heat-stable inhibitor(s) of liver uroporphyrinogen decarboxylase was extractable from uroporphyric livers. Although oxidation of uroporphyrinogen I to uroporphyrin I by hepatic microsomes from rats pretreated with porphyrogenic regimes of HCB and PCBs was induced, there was no correlation with the in vivo sex difference in porphyria development. Levels of total cytochrome P450 and pentoxyresorufin and benzyloxyresorufin dealkylase activities (associated with cytochrome P450IIB1) were greater in microsomes from control, HCB, PCB and PBB treated male rats than females. In contrast, ethoxyresorufin deethylase activity (associated with cytochrome P450IA1) was always significantly greater in females. These findings were confirmed by immunoblotting with polyclonal antibodies to cytochromes P450IA1, IA2 and IIB1. Immunocytochemical studies showed that, even after 30 weeks of HCB exposure, cytochromes P450IA1 and P450IA2 were still more highly induced in female liver, especially in the centrilobular region. The results are consistent with the association of cytochrome P450IA isoenzymes with uroporphyria development, although the sex difference in P450IA levels alone may not be marked enough to provide the complete explanation for the pronounced susceptibility of females to HCB.

Inducible bilirubin-degrading system in the microsomal fraction of rat liver.

The hypothesis that treatment of Gunn rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates an alternative pathway of bilirubin disposal, involving an induced form of cytochrome P-450 [Proc. Natl. Acad. Sci. USA 75:682-685 (1978)], has been investigated by studying the mechanisms of bilirubin oxidation in chemical model systems and in liver microsomal systems in vitro. Hematin, copper sulfate, and the iron chelate of EDTA were all active in promoting degradation of bilirubin in the presence of hydrogen peroxide. Evidence was obtained for a microsomal bilirubin-degrading system that could be induced in the liver by treating either rats or chick embryos with TCDD, beta-naphthoflavone, or 3,4,3',4'-tetrachlorobiphenyl (3,4-TCB) in vivo. The activity of this system required NADPH and oxygen and was markedly stimulated by addition of 3,4-TCB (a planar polyhalogenated biphenyl) and much less markedly by the nonplanar analogue 2,4,2',4'-tetrachlorobiphenyl. These two biphenyls were also inhibitory towards the 7-ethoxyresorufin O-deethylase activity of the induced microsomes and here again the nonplanar analogue was markedly less active. Dose-response experiments for stimulation of bilirubin breakdown and inhibition of 7-ethoxyresorufin O-deethylase activity after addition of 3,4-TCB in vitro showed both effects to be caused by similar concentrations of the biphenyl. These results suggest that a polyhalogenated chemical may interact with TCDD-induced microsomes, inhibit their monooxygenase activity, and stimulate production of a bilirubin-degrading species.

Nephrotoxicity of Penicillium aurantiogriseum, a possible factor in the aetiology of Balkan endemic nephropathy.

Water-soluble components of a nephrotoxic isolate of Penicillium aurantiogriseum have been fractionated by sequential ion-exchange, size-exclusion gel filtration, reverse-phase silica chromatography and HPLC. Nephrotoxicity in the rat was confined to a size-exclusion fraction approximating to 1,500 daltons, which also inhibited DNA synthesis in cultured kidney cells. The more sensitive in vitro assay allowed toxicity to be followed to a sub-fraction from gradient-elution HPLC which in further HPLC resolved into a small group of glycopeptides. Recent Yugoslavian P. aurantiogriseum isolates, from a village in which the idiopathic human disease Balkan Nephropathy is hyperendemic, elicited a similar nephropathology and were acutely cytotoxic, reinforcing a need to regard this novel Penicillium nephrotoxin as a potential factor in human nephropathy.

Pleiotropic effect of the gene hairless on hepatotoxicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in mice.

Treatment of mice of the A2G-hr/+ congenic line with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in the development of hepatic porphyria over a period of 4 weeks. Female mice responded to a lesser extent than did males. The degree of porphyria in haired heterozygotes (hr/+) was less than in the corresponding hairless homozygotes (hr/hr) and the haired mice had lower resting metabolic rates than hairless mice. Adaptation of mice of either genotype to a 32-33 degrees C environment resulted in a decrease in resting metabolic rate and a reduction in hepatic porphyrin levels. Histologically-demonstrated necrotic changes in livers were accompanied by increased activity of alanine aminotransferase and sorbitol dehydrogenase in the plasma; however, there was no clear temporal trend in plasma enzyme levels. Elevated environmental temperature reduced the plasma alanine aminotransferase activity. The study provided evidence for a pleiotropic effect of variation at the hr locus being expressed in TCDD hepatotoxicity. Suggestions for mechanisms whereby the effect can be mediated through alterations in resting metabolic rate are made.

Mechanistic studies of the inhibition of hepatic uroporphyrinogen decarboxylase in C57BL/10 mice by iron-hexachlorobenzene synergism.

Porphyria was induced in C57BL/10 mice with iron overload by a single oral dose (100 mg/kg) of hexachlorobenzene (HCB). Within 2 weeks hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was inhibited, reaching a maximum (greater than 95%) at 6-8 weeks. There was no recovery by 14 weeks, despite a fall in liver HCB concentrations to only 6% of the day-3 value. The major rise in hepatic porphyrin levels occurred after 4 weeks and secondary inhibition of uroporphyrinogen synthase (EC 4.2.1.75) was inferred from the progressively greater proportion of uroporphyrin I present relative to the III isomer. Plasma alanine aminotransferase (EC 2.6.1.2) activity was also elevated. Although, in further studies, total microsomal cytochrome P-450 content and ethoxyphenoxazone de-ethylase activity reached a peak a few days after dosing and had declined significantly at the time of maximum inhibition of the decarboxylase, additional treatment of HCB-dosed mice with a cytochrome P1-450 inducer, beta-naphthoflavone, enhanced the inhibition, whereas piperonyl butoxide, an inhibitor of cytochrome P-450, partially protected. Uroporphyrinogen decarboxylase was not radiolabelled in vivo by [14C]HCB. There was no major difference in the ability to hydroxylate HCB between hepatic microsomes from induced C57BL/10 mice and those from the insensitive DBA/2 strain. By contrast, lipid peroxidation, in the presence of NADPH, was 8-fold greater in control C57BL/10 microsomes than in DBA/2 microsomes and was stimulated by iron treatment (although not by HCB). The results suggest that the inhibition of hepatic uroporphyrinogen decarboxylase is unlikely to be due to a direct effect of a metabolite of HCB but to another process requiring a specific cytochrome P-450 isoenzyme and an unknown iron species.

Continued depression of hepatic uroporphyrinogen decarboxylase activity caused by hexachlorobenzene or 2,3,7,8-tetrachlorodibenzo-p-dioxin despite regeneration after partial hepatectomy.

Hepatic uroporphyrinogen decarboxylase activity in male C57BL/10 mice was maintained in regenerated liver after recovery from two-thirds hepatectomy. In contrast, there was little increase in enzyme activity in regenerated liver from animals previously treated with hexachlorobenzene (HCB) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These chemicals initially cause depression of uroporphyrinogen decarboxylase activity over a time much longer than the period allowed for regeneration. Estimation of HCB levels showed that there was only a small amount of redistribution to the liver during regrowth. The results demonstrate that HCB and TCDD induce either formation of a toxic metabolite or some other inhibitory process and that this can be sustained for a long period which delays recovery to the normal state.

An investigation of genetic variation within a series of congenic strains of mice.

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.

Incomplete correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin hepatotoxicity with Ah phenotype in mice.

Pretreatment of male mice of the inbred strains A2G, BALB/c, C57BL/10, and AKR with iron dextran synergized the action of a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 75 micrograms/kg) in causing hepatic porphyria and necrosis 35 days later. There was no effect in DBA/2 mice. Increased porphyrin levels were associated with decreased hepatic activity of uroporphyrinogen decarboxylase. Iron alone had no effect on porphyrin levels or decarboxylase activity. In male BALB/c mice given TCDD alone there was a delay in the onset of porphyria. Female BALB/c, AKR, and AKR X DBA/2 F1 mice were more resistant to the porphyrinogenic effect of TCDD than males. Development of porphyria did not correlate with Ah phenotype of the mice. The inheritance of sensitivity to TCDD in crosses of the AKR and DBA/2 strains, both Ah nonresponsive, was studied by a biometrical genetic analysis. The inheritances of increased porphyrin levels and of increased plasma activity of enzymes indicative of hepatic necrosis were both complex. Segregation of alleles at more than one locus was required to explain the data. A lack of correlation of porphyrins with plasma enzyme levels in the F2 generation suggested that the expression of these traits was determined independently. Genes other than Ah influence the development of TCDD-induced hepatotoxicity in mice.

Biochemical and morphological changes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat liver cell plasma membrane.

The administration of single oral doses of 200 micrograms kg-1 of 2,3,7,8-tetrachlorodibenzo-p-dioxin to females of the outbred, Lac : P strain of rat results in the formation of hepatic multinucleate cells by cell fusion. Liver cell plasma membranes isolated 6 or 11 days after dosing show two distinct changes. The first is a decrease of the activity of K+-Mg2+-ATPase, which confirms histochemical observations. The second is the formation, in those animals showing a more severe intoxication, of a population of plasma membranes which are less dense than usual and which consist of extended membrane sheets. It is suggested that these alterations are consequent on a disturbance of lipid metabolism in the hepatic cells and that they represent early manifestations of the toxic process which leads to the formation of multinucleate cells.

Balkan (endemic) nephropathy.

The clinical and pathological features of Balkan (endemic) nephropathy are discussed and correlations of incidence with excess late summer and autumn rainfall outlined. Cultures of a strain of Penicillium verrucosum var. cyclopium isolated from maize collected in an endemic area were fed to rats and lesions were produced in the straight third segment of the proximal kidney tubules. Extensive degeneration and nuclear changes were seen and on prolonged feeding further nuclear enlargement (to greater than 6n) and the formation of multinucleate cells occurred. The relevance of these findings to the clinical disease in man, especially the occurrence of urinary tract tumours, and the evidence supporting mycotoxin involvement, are discussed.

Different patterns of hepatic microsomal enzyme activity produced by administration of pure hexachlorobiphenyl isomers and hexachlorobenzene.

Three hexachlorobiphenyl isomers, 2,2',4,4',5,5'-hexachlorobiphenyl (I), 2,2',3,3',4,4'-hexachlorobiphenyl (II) and 2,2',3,4,4',5'-hexachlorobiphenyl (III), have been administered to rats and the effects of these three compounds upon hepatic microsomal drug metabolism and upon hepatic porphyrins have been studied. Comparisons have been made with hexachlorobenzen and a commercial polychlorinated biphenyl mixture, Aroclor 1254. From measurements of activities of microsomal drug oxidations in vitro, the durations of pharmacological actions of certain drugs in vivo and spectral shifts associated with cytochrome P-450 it is shown that the three pure hexachlorobiphenyl isomers initially produce changes in hepatic microsomal activity which resemble those seen after treatment with phenobarbitone (PB). In contrast, following chronic feeding of the isomers, compounds II and III but not I produce a pattern of hepatic microsomal enzyme activity which shows some characteristics of the 3-methylcholanthene (3-MC) and some characteristics of the phenobarbitone classes of inducer. Also, compounds II and III, but not I, cause accumulation in the liver of porphyrins containing either seven or eight carboxyl groups. These two responses are similar to those observed following hexachlorobenzene treatment and suggest that a relationship may exist between the mixed pattern of enzyme induction and the onset of hepatic porphyrin accumulation.