Organochlorine chemicals in seafood: occurrence and health concerns.

The cheap availability of chlorine gas, together with the development of industrial chlorinating procedures in the 20th century, led to the production of a wide range of organochlorine compounds many with a variety of commercial applications, including usage as insecticides and defoliants and polychlorinated biphenyls (PCBs) used as coolants in electricity supply transformers. However, it was soon found that many of these technologically valuable chemicals suffered from a major disadvantage in that they resisted biodegradation and that the continued use of these compounds would lead to their persistence and accumulation in the environment and thus enter the human food chain. Despite regulatory bans or strict limits on usage being imposed on organochlorine pesticides in most countries, these compounds continue to be detected in measurable amounts in the eco-system including marine life. In general, organochlorine levels in fish intended for human consumption are low and probably below levels likely to adversely affect human health. Populations at higher risk than most people are those subsisting largely on fish and other marine life. Additionally, fish oils obtained from contaminated fish, if consumed in substantial quantities by infants and young children, might present potential health problems if levels are not continually regulated. Behavioral and neurological effects have been reported in children and ascribed to the consumption of PCB contaminated diet including fish. Another current major human health concern, yet to be resolved, about organochlorine contaminants in the human diet relates to the potential ability of many of these chemicals at low doses to act as "endocrine disruptors".

Nitrate, nitrite and N-nitroso compounds.

A risk assessment has been made on nitrate, nitrite and N-nitroso compounds encountered in the human diet. Vegetables constitute a major source of nitrate providing over 85% of the average daily human dietary intake. Nitrite and N-nitroso compounds present in the diet contribute relatively small amounts to the body burden and the major source of these biologically reactive compounds is derived from the bacterial and mammalian metabolism of ingested nitrate. Additionally, endogenous synthesis provides an important source contributing to the body burden of nitrate. Data from animal toxicological studies, human effects and epidemiological surveys have been reviewed and evaluated. It is concluded that there is no firm scientific evidence at present to recommend drastic reductions beyond the average levels of nitrate encountered in vegetables grown in keeping with good agricultural practice. Recommendations have also been made for further animal and human studies to be carried out to elucidate the potential risks to man from ingested nitrate.

A 28-day feeding study with ethyl acetoacetate in rats.

Ethyl acetoacetate encapsulated in gum arabic was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 100, 300 and 1000 mg/kg body weight/day. A further group of 16 male and 16 female rats was given rodent diet containing gum arabic as a control. The administration of ethyl acetoacetate in the diet did not adversely affect the growth or general health of the animals or their food intakes. None of the minor variations observed in the haematology, serum chemical analyses or urine analyses are considered to be indicative of a treatment-related toxic effect. Caecal enlargement was seen in male rats treated with the top dose of ethyl acetoacetate, but this was accompanied by a normal histopathology. Few histopathological abnormalities were observed. Proteinaceous casts were found in the bladder of approximately half the male rats given 1000 mg ethyl acetoacetate/kg, and nephrocalcinosis was a common occurrence in female rats in this dose group. Renal function was unimpaired in treated male and female rats, and the histopathological findings are common in the strain of rats chosen for this study. Although the caecal enlargement and the changes in kidney and bladder of rats given 1000 mg ethyl acetoacetate/kg are noted, it is considered that ethyl acetoacetate did not produce treatment-related adverse effects in rats during this study.

A 28-day feeding study with methyl isoeugenol in rats.

Methyl isoeugenol was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 30, 100 and 300 mg/kg body weight/day. A further group of 16 male and 16 female rats was given the rodent diet as a control. The administration of methyl isoeugenol in the diet did not adversely affect the growth or general health of the animals or their food intakes. Although high dose animals of both sexes had increased lymphocyte and total white blood cell counts, these are not considered, in isolation, to be an adverse effect of treatment. None of the minor variations observed in the serum chemical analyses or urine analyses is considered to be indicative of a treatment-related toxic effect. An increase in liver weight, adjusted for body weight, was seen in male and female rats receiving 300 mg methyl isoeugenol/kg body weight. Few histopathological abnormalities were observed. Although the incidence of kidney and Harderian gland lesions was higher for high dose animals compared with the controls, the lesions are of a type that occurs spontaneously and are thus not considered to be attributable to treatment with methyl isoeugenol. While the increased liver weight and white blood cell counts of rats given 300 mg methyl isoeugenol/kg body weight may represent effects of treatment, it is not considered that there is any reason to regard these as adverse effects.

Comparison of the metabolism and disposition of [3-14C]coumarin in the rat and marmoset (Callithrix jacchus).

Male Sprague-Dawley rats and marmosets were given a single oral 25 mg/kg dose of [3-14C]coumarin and the excretion of radioactivity in the expired air, urine and faeces monitored up to 96 h. Excretion profiles were similar in both species with the bulk of the dose being excreted in the urine and faeces within 24 h. Chromatographic analysis of 0-48 h urine samples revealed similar metabolic profiles with only small amounts of unchanged coumarin and very little 7-hydroxycoumarin. Coumarin 7-hydroxylase activity was not detectable in hepatic microsomes from either species. These results demonstrate that the disposition of [3-14C]coumarin was similar in the rat and marmoset, a New World primate, and that both species, unlike man, are poor 7-hydroxylators of coumarin.

Short-term toxicity study of Green S in rats.

Groups of 15 rats of each sex were fed Green S at dietary concentrations to provide dose levels of 0 (control), 250, 500 or 1500 mg/kg body weight/day for 13 wk. Additional groups of five animals of each sex were given the same treatments for 2 or 6 wk. There was a marked excretion of green colour in the faeces and some green colouring of the urine, although the latter may have been due to contamination. The males showed increased water and food intakes associated, particularly at the highest dose, with a higher rate of body-weight gain. Haematological examination revealed a transitory mild anaemia at the highest dose level, whilst no findings indicative of a toxic effect were found in the renal concentration tests or the serum analyses. With a dose of 1500 mg Green S/kg a greater proportion of the rats showed higher urinary protein, protein casts, increased caecal weight, thyroid degeneration in female animals and enlargement of the lymph nodes in the intestine wall. The no-effect dose level for Green S in this study was considered to be 500 mg/kg.

Structure-activity relationships for induction of peroxisomal enzyme activities by phthalate monoesters in primary rat hepatocyte cultures.

Rat hepatocytes were cultured for 70 hours with a series of four isomeric octyl and five isomeric hexyl phthalate monoesters, and their effects on peroxisomal fatty acid beta-oxidation (palmitoyl-CoA oxidation) and carnitine acetyltransferase activities determined. All nine monoesters produced dose-related increases in enzyme activities and marked quantitative compound potency differences were observed. Generally octyl isomers were more potent than hexyl isomers and 2- and 3-ethyl substituted isomers were more potent than their straight chain and 1-ethyl substituted analogs. For example, mono(2-ethylhexyl)phthalate was more potent than mono(1-ethylhexyl)phthalate, and this was also observed after oral administration of the two isomers to rats for seven days. The cell culture data for induction of palmitoyl-CoA oxidation were used to generate quantitative structure-activity relationships. Relatively poor correlations were observed between biological activity and simple hydrophobic parameters, but a good correlation was obtained when compound electronic structural parameters, obtained by molecular orbital calculations, were employed. These studies demonstrate relationships between biological activity and chemical structure for a series of phthalate monoesters and indicate the potential usefulness of primary rat hepatocyte cultures to screen compounds for peroxisome proliferation.

Effect of prolonged administration of clofibric acid and di-(2-ethylhexyl)phthalate on hepatic enzyme activities and lipid peroxidation in the rat.

Male Sprague-Dawley rats were fed diets containing either 0.5% clofibric acid (CA) or 2% di-(2-ethylhexyl)phthalate (DEHP) for 2 years. Both compounds produced liver enlargement which was accompanied by the formation of liver nodules. Hepatic peroxisomal and microsomal fatty acid oxidising enzyme activities were induced in both large nodules and host tissue (i.e. tissue remaining after removal of large nodules) preparations from CA and DEHP treated rats. In contrast, little change in catalase activity was observed and the activities of cytosolic GSH peroxidase and GSH S-transferases were markedly reduced. Increased lipid peroxidation was observed by measurement of conjugated dienes in host tissue homogenates from CA and DEHP treated rats. Microsomal NADPH-dependent lipid peroxidation was also stimulated. Histological examination revealed extensive lipofuscin deposition in non-nodular, but not in nodular, tissue sections from treated rats. These results demonstrate that prolonged peroxisome proliferation can result in lipid peroxidation and that certain enzymes which metabolise hydrogen peroxide and organic hydroperoxides are either little affected or markedly inhibited.

Increased incidence of abnormal foetuses in the offspring of cyclophosphamide-treated male mice.

The incidence of morphologically abnormal foetuses in the litters of cyclophosphamide (CP)-treated male mice was investigated and compared with control values. In two experiments (100 mg/kg) CP was shown to increase the incidence of grossly abnormal foetuses over that seen in the controls, although neither was statistically significant in isolation. When the probabilities from the two tests of significance were combined using the method of Fisher the result was significant (P = 0.02). These results suggest that an acute exposure to a mutagen in male mice can cause genetic damage that results in an increased incidence of phenotypically abnormal offspring. However, the large numbers of animals required and the variable control level of abnormalities, indicate that this dosing regimen is an inefficient method of studying the genetic mechanisms responsible for the effects seen.

An evaluation of the decision tree approach for assessing priorities for safety testing of food additives.

In this publication we report an evaluation of the decision tree scheme of Cramer, Ford and Hall (1978) for assigning priorities for toxicity testing of chemicals. The original scheme has been modified to allow more chemical structures to be considered and to take into account recent advances in toxicology. The majority of the food additives permitted in either the UK, USA or Canada have been processed through the modified decision tree questionnaire and their classification compared with currently available chronic toxicity data. A large proportion of the additives (53/73) assigned to the lowest toxicity (I) class have a low order of chronic oral toxicity as do many of the compounds assigned to the moderate toxicity (II) class. Although the majority of the additives assigned to the highest toxicity (III) class are substantially more toxic than those in the lower toxicity classes, some relatively innocuous compounds reached this classification. In addition, a few toxic compounds were assigned to the lowest toxicity class. The reasons for these incorrect assignments are discussed. It was concluded that the decision tree approach, although less discriminating than originally suggested, remains a useful method for classifying compounds in terms of their probable toxicity and that further modifications to the tree could be made.

Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males.

Adult male CD rats and CD-1 mice were given a single oral dose of ethylene glycol monomethyl ether (EGM) at 0, 500, 750, 1,000, or 1500 mg/kg. Groups of 10 were killed at weekly intervals after dosing for analysis of sperm counts and morphology or testicular histology; further groups of 10 were sequentially mated to pairs of virgin females to test for dominant lethality or gross foetal malformations in the F1 generation (F1 abnormalities). EGM was found to deplete the spermatocytes of both species severely, principally pachytene cells, but with other stages affected with increasing dose. A delay in the progression of spermatogenesis may account for a discrepancy between the apparent stage-specificity of damage deduced from lowered sperm counts and that observed histologically. In the rat, morphological abnormalities were observed in sperm that had been exposed as spermatocytes; in the mouse, however, the sensitive cells were the late spermatocytes and early spermatids. In all these parameters there was an indication of a dose-response relationship in both rats and mice. In the mating studies EGM induced a dose-related decrease in fertility 5 weeks after dosing in the rat, but complete sterility in all but the lowest dose after 6 weeks. In contrast, EGM had no effect on the reproductive capacity of the mouse. There was no statistically significant evidence for the induction of dominant lethal mutations or F1 abnormalities in either species. A single oral dose of cyclophosphamide (CTX) at 100 mg/kg induced a significant increase in dominant lethality in both species. CTX reduced the number of total implants in the rat and induced a nonsignificant increase in the number of abnormal offspring sired by treated male mice.

The histopathology and biochemistry of phenobarbitone-induced liver nodules in C3H mice.

The sequential development of hepatic nodules induced by phenobarbitone (PB) has been studied in the C3H/He strain of mouse, a strain prone to the development of spontaneous liver tumours. PB was administered in the diet to young male animals at a dose level of 85 mg/kg/day for up to 91 weeks. Control and PB-treated mice developed hepatic nodules within 60 weeks. In control animals the nodules consisted of small basophilic cells, and by 80 weeks a small proportion of the animals had developed unequivocal hepatocellular carcinoma. The basophilic nodules were similar in many respects to those induced by N-nitrosodiethylamine. In PB-treated mice the incidence of basophilic nodules was similar to that in controls. These animals also developed hepatic nodules formed of large eosinophilic cells which were readily dissectable from the surrounding host tissue by 60 weeks. Biochemical investigations into the large eosinophilic nodules from PB treated mice showed that both Phase I and Phase II types of xenobiotic metabolizing enzyme activities were induced to levels either similar to, or greater than in the surrounding host tissue. In contrast, enzyme activities in basophilic nodules from untreated mice killed at 91 weeks, were generally similar to or lower than in the surrounding host tissue. The presence of eosinophilic nodules did not lead to an increase in the incidence of hepatocellular carcinoma in the PB-treated C3H/He mice. Concurrent experiments conducted in the C57BL/6 strain of mouse did not result in the development of liver nodules at 60 weeks. Thus, the eosinophilic nodules induced by phenobarbitone in C3H/He mice appear to be distinctly different from the basophilic liver nodules arising spontaneously or to basophilic nodules produced by the hepatocellular carcinogen N-nitrosodiethylamine.

Hepatic effects of phthalate esters and related compounds--in vivo and in vitro correlations.

The hepatic effects of phthalate esters and related compounds on peroxisomal and microsomal enzyme activities were investigated in the intact animal and in primary hepatocyte cultures. In vitro studies with a series of phthalate monoesters demonstrated structure activity differences in the induction of the specific peroxisomal marker KCN-insensitive palmitoyl-CoA oxidation and also of carnitine acetyltransferase. These effects could be reproduced in vivo, and potency differences were also observed between di(2-ethylhexyl) phthalate (DEHP) and its straight-chain isomer, di-n-octyl phthalate. In in vivo studies, DEHP, mono(2-ethylhexyl) phthalate, and clofibrate/clofibric acid produced large increases in liver size and peroxisomal enzyme activities in Sprague-Dawley rats and Chinese hamsters, but had less effect in Syrian hamsters. These effects could be largely reproduced in vitro where good responses were obtained with rat and Chinese hamster hepatocytes, but either little or no effect with Syrian hamster and Dunkin-Hartley guinea pig hepatocytes. Both DEHP and clofibrate appeared to induce similar form(s) of microsomal cytochrome P-450 which exhibited a high specificity towards lauric acid hydroxylation. With a range of hypolipidemic agents, including phthalate monoesters, a good correlation was observed between the induction of peroxisomal and microsomal enzyme activities in rat hepatocyte cultures. These results thus demonstrate a good relationship between the in vivo and in vitro effects of phthalate esters and related compounds and suggest that hepatocyte cultures may be useful for further studies on the hepatic effects of peroxisome proliferators.

Influence of phagocyte-derived active oxygen species in tissue responses to tumour promoters and irritants.

In response to stimuli such as irritant chemicals, inorganic particles or micro-organisms, phagocytic cells produce a variety of chemical species including superoxide and hydrogen peroxide. These reactive oxygen species are cytotoxic and genotoxic to cultured cells and may be responsible for some of the tissue damage associated with inflammation in vivo. The role of phagocyte-derived oxygen radicals in the effects of irritants, tumour promoters and carcinogenic inorganic materials has been explored using mixed cultures of phagocytes and fibroblasts. Catalase-inhibitable cytotoxicity and genetic damage were demonstrated in fibroblasts exposed to neutrophils stimulated with the irritant and tumour promoter phorbol myristate acetate or with zymosan particles. To investigate this phenomenon further, other cell systems have been developed, using epidermal keratinocytes or mesothelial cells in combination with neutrophils or macrophages.

Studies on the mechanism of diet-induced nephrocalcinosis: calcium and phosphorus metabolism in the female rat.

The disposition of calcium and phosphorus in female Sprague-Dawley rats fed either a low- or a high-calcium semi-synthetic diet for up to 9 wk from weaning has been investigated. The rats fed the low-calcium diet (calcium to phosphorus ratio approximately 1:1) developed cortico-medullary nephrocalcinosis within 6 wk, whereas those fed the high-calcium diet (calcium to phosphorus ratio greater than 1.5:1) did not develop any lesion during this time. Intake and excretion of calcium was greater at all times in the animals fed the high-calcium diet than in those on the low-calcium diet. However, the true absorption and retention of calcium was not significantly different between the two diet groups. Phosphorus intake and total excretion were similar for both groups of animals, although urinary excretion accounted for less than 1% of the total in the animals fed the high-calcium diet but more than 50% in those fed the low-calcium diet. Bioavailability of phosphorus decreased with age in both groups. Recovery of injected 45Ca in urine and faeces increased during the experimental period from approximately 3 to 11% of the dose in rats on the low-calcium diet and from approximately 9 to 13% in the high-calcium group. Recovery of both injected and orally administered 32P was substantially greater in the low-calcium group than in the high-calcium group at wk 3 and 5, but at wk 9 was only greater after intramuscular administration. At the earlier times the urine was the major route of 32P excretion. It appears that no gross disturbances of calcium or phosphorus metabolism occurred in female rats maintained on a diet that resulted in the rapid development of kidney calcification.

Aspects of the testicular toxicity of phthalate esters.

Di(2-ethylhexyl) phthalate (DEHP) produced seminiferous tubular atrophy and reductions in seminal vesicle and prostate weight in 4-week-old, but not in 15-week-old rats. Di-n-pentyl phthalate (DPP) did produce atrophy in the older rats but this developed more slowly than in young animals. Coadministration of testosterone or gonadotrophins did not protect against phthalate-induced testicular toxicity but did partly reverse the depression of seminal vesicle and prostate weight. Secretion of seminiferous tubule fluid and androgen binding protein by the Sertoli cells was markedly suppressed within 1 hr of a dose of DPP or mono-2-ethylhexyl phthalate (MEHP) in immature rats. This occurred less rapidly in mature rats. [14C]Mono-n-pentyl phthalate and [14C]MEHP penetrated the blood testis barrier only to a very limited extent. These findings and the early morphological changes in the Sertoli cells produced by DPP suggest that phthalate esters may act initially to cause Sertoli cell injury, the subsequent loss of germ cells occurring as a consequence of this. Some features of the testicular lesion could be reproduced in primary cocultures of rat Sertoli and germ cells. Structure activity studies with a range of phthalate monoesters showed good agreement between the induction of germ cell detachment in culture and testicular toxicity in vivo. Three metabolites of MEHP (metabolites V, VI, and IX) were much less toxic in culture than MEHP itself, suggesting that the latter may be the active testicular toxin from DEHP.

Studies on the genetic effects of phthalic acid esters on cells in culture.

Mono(2-ethylhexyl) phthalate (MEHP) induced chromosome aberrations in cells of two culture lines, one derived from Chinese hamster ovary cells (CHO) and the other from rat liver cells (RL4). In CHO cells, the clastogenicity of MEHP was unaffected by the presence of an exogenous metabolic activation system (S-9 mix). 2-Ethylhexanol, o-phthalic acid, and phthalic anhydride were without effect. Cytochemical methods and assays for carnitine acetyltransferase and KCN-insensitive palmitoyl CoA oxidation were employed to determine whether chromosome damage was associated with peroxisome proliferation. No evidence of an increase in peroxisome numbers or of induction of marker enzymes was found in CHO cells treated with MEHP for up to 72 hr. Clofibric acid and BR931 were also ineffective. Observations on changes in CHO cell structure and permeability, and on the haemolytic effects of phthalate monoesters, suggest that the cytotoxicity of MEHP may be due primarily to its action on cell membranes. Since chromosome damage was observed only at cytotoxic concentrations, it is suggested that damage to lysosomal membranes and the release of endonucleases may be responsible for the observed clastogenicity of MEHP in vitro.

Structure activity studies on the induction of peroxisomal enzyme activities by a series of phthalate monoesters in primary rat hepatocyte cultures.

A series of 9 phthalate monoesters were cultured with primary rat hepatocytes for 70 h and their effect on peroxisomal fatty acid beta-oxidation (palmitoyl-CoA oxidation) determined. Marked quantitative differences in the induction of enzyme activity were observed with both alkyl chain length and position of side chain substitution affecting compound potency. With all 9 compounds a good correlation was observed between electronic structural parameters obtained by molecular orbital calculations and biological activity in the cell culture system. These results demonstrate a relationship between chemical structure and biological activity for a series of phthalate monoesters and indicate the potential usefulness of primary hepatocyte cultures to screen compounds for peroxisome proliferation.

Malformations induced in cultured rat embryos by enzymically generated active oxygen species.

Day 9.5 rat embryos were exposed in culture to xanthine/xanthine oxidase generated active oxygen species. Growth and development were assessed after 46 hr of culture. The treatment induced abnormalities of the neural suture, the severity of which increased in a dose-related manner with the concentration of substrate or enzyme. Glutathione (10 mM) or catalase (50 micrograms/ml) either partially or completely abolished the effects of xanthine/xanthine oxidase, whereas the addition of superoxide dismutase (50 micrograms/ml) or desferrioxamine (1mM) did not reduce the number of malformed embryos. These findings suggest that hydrogen peroxide and/or hydroxyl radicals are responsible for the effects of xanthine and xanthine oxidase.