Joseph G. Vos

Health Effects of Endocrine-Disrupting Chemicals on Wildlife, with Special Reference to the European Situation

Many wildlife species may be exposed to biologically active concentrations of endocrine-disrupting chemicals. There is strong evidence obtained from laboratory studies showing the potential of several environmental chemicals to cause endocrine disruption at environmentally realistic exposure levels. In wildlife populations, associations have been reported between reproductive and developmental effects and endocrine-disrupting chemicals. In the aquatic environment, effects have been observed in mammals, birds, reptiles, fish, and mollusks from Europe, North America, and other areas. The observed abnormalities vary from subtle changes to permanent alterations, including disturbed sex differentiation with feminized or masculinized sex organs, changed sexual behavior, and altered immune function. For most reported effects in wildlife, however, the evidence for a causal link with endocrine disruption is weak or nonexist-ing. Crucial in establishing causal evidence for chemical-induced wildlife effects appeared semifield or laboratory studies using the wildlife species of concern. Impaired reproduction and development causally linked to endocrine-disrupting chemicals are well documented in a number of species and have resulted in local or regional population changes. These include: • Masculinization (imposex) in female marine snails by tributyltin, a biocide used in antifouling paints, is probably the clearest case of endocrine disruption caused by an environmental chemical. The dogwhelk is particularly sensitive, and imposex has resulted in decline or extinction of local populations worldwide, including coastal areas all over Europe and the open North Sea. • DDE-induced egg-shell thinning in birds has caused severe population declines in a number of raptor species in Europe and North America. • Endocrine-disrupting chemicals have adversely affected a variety of fish species. In the vicinity of certain sources (e.g., effluents of water treatment plants) and in the most contami nated areas is this exposure causally linked with the effects on reproductive organs that could have implications for fish populations. However, there is also a more widespread occurrence of endocrine disruption in fish in the U.K., where estrogenic effects have been demonstrated in freshwater systems, in estuaries, and in coastal areas. • In mammals, the best evidence comes from the field studies on Baltic gray and ringed seals, and from the Dutch semifield studies on harbor seals, where both reproduction and immune functions have been impaired by PCBs in the food chain. Reproduction effects resulted in population declines, whereas impaired immune function has likely contributed to the mass mortalities due to morbillivirus infections. • Distorted sex organ development and function in alligators has been related to a major pesticide spill into a lake in Florida, U.S.A. The observed estrogenic/antiandrogenic effects in this reptile have been causally linked in experimental studies with alligator eggs to the DDT complex. Although most observed effects currently reported concern heavily polluted areas, endocrine disruption is a potential global problem. This is exemplified by the widespread occurrence of imposex in marine snails and the recent findings of high levels of persistent potential endocrine-disrupting chemicals in several marine mammalian species inhabiting oceanic waters.

Contaminant-induced immunotoxicity in harbour seals: wildlife at risk?

Persistent, lipophilic polyhalogenated aromatic hydrocarbons (PHAHs) accumulate readily in the aquatic food chain and are found in high concentrations in seals and other marine mammals. Recent mass mortalities among several marine mammal populations have been attributed to infection by morbilliviruses, but a contributing role for immunotoxic PHAHs, including the polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) was not ruled out. We addressed this issue by carrying out a semi-field study in which captive harbour seals were fed herring from either the relatively uncontaminated Atlantic Ocean or the contaminated Baltic Sea for 2 years. We present here an overview of results obtained during this study. An impairment of natural killer (NK) cell activity, in vitro T-lymphocyte function, antigen-specific in vitro lymphocyte proliferative responses, and in vivo delayed-type hypersensitivity and antibody responses to ovalbumin was observed in the seals fed the contaminated Baltic herring. Additional feeding studies in PVG rats using the same herring batches suggested that an effect at the level of the thymus may be responsible for changes in cellular immunity, that virus-specific immune responses may be impaired, and that perinatal exposure to environmental contaminants represents a greater immunotoxic threat than exposure as a juvenile or adult. Together with the pattern of TCDD toxic equivalents of different PHAHs in the herring, these data indicate that present levels of PCBs in the aquatic food chain are immunotoxic to mammals. A review of contaminant levels in free-ranging harbour seals inhabiting polluted areas of Europe and North America suggests that many populations may be at risk to immunotoxicity. This could result in diminished host resistance and an increased incidence and severity of infectious disease.

Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated naphthalene in two commercial polychlorinated biphenyls.

In a previous study (Vos & Koeman, Toxic. appl. Pharmac. 1970. In press) a significant difference in toxicity was found between three commercial polychlorinated biphenyl (PCB) preparations, high mortality, liver necrosis and chick oedema-like lesions being associated with only two of the compounds tested. In the present study, column and gas chromatography demonstrated the presence of polar compounds in the 25% diethyl ether fraction of each of these two PCB preparations, and a chick-embryo assay confirmed the high toxicity of this fraction. The polar compounds were not found in the corresponding fraction of the third PCB preparation; neither did this preparation have a significantly toxic effect on the chick embryo. Subsequent mass spectrometric and microcoulometric analyses indicated the identity of the polar compounds, which included tetra- and pentachlorodibenzofuran. The significance of these impurities is discussed. It is concluded that evaluation of animal tests on PCB is made extremely difficult by the presence of chlorinated dibenzofurans in some commercial PCB preparations.

Toxicity of bis(tri-n-butyltin)oxide in the rat: II. Suppression of thymus-dependent immune responses and of parameters of nonspecific resistance after short-term exposure

To evaluate the functional significance of bis(tri-n-butyltin)oxide (TBTO)-induced thymus atrophy, lymphocyte depletion in spleen and lymph nodes, lymphopenia, and increased serum IgM and decreased IgG concentrations, in vivo and in vitro function studies were performed for specific and nonspecific resistance. Weaned male rats were fed diets containing 0, 20, or 80 mg TBTO/kg for a least 6 weeks. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin as well as tuberculin were significantly depressed at both dietary concentrations. Resistance to the nematode Trichinella spiralis was significantly suppressed as shown by a retarded expulsion of adult worms from the small intestine, increased counts of muscle larvae, reduced inflammatory reaction in parasitized musculature, and suppressed serum IgE titers. Also the secondary mercaptoethanol-resistant (presumably IgG) hemagglutinating antibody titer to sheep red blood cells was significantly reduced, while no significant alterations were found in IgM and IgG titers to T. spiralis, ovalbumin, and tetanus toxoid. TBTO exposure reduced the response of thymocytes in both treatment groups and of spleen cells in the 80-mg/kg group upon stimulation with T-cell mitogens and increased the response of spleen cells to B-cell mitogens. When calculated per whole spleen, the response to T-cell mitogens was strongly impaired but unaltered by B-cell mitogens. This difference can be explained by a relative increase of splenic B cells as a result of reduced numbers of T cells, as shown by cell surface marker analysis using monoclonal antibodies. Reduced splenic T-cell numbers appeared equally due to a decreased number of T helper and to T suppressor cells. From these data and from results of a time-sequence study in which effects of TBTO on cell count and cell viability of thymus, spleen, and bone marrow were investigated, it is concluded that TBTO-induced immunodeficiency was primarily due to its direct toxic action on thymocytes. When cultured in vitro in the presence of TBTO, viability of thymus and bone marrow cells was equally reduced, while after in vivo treatment viability of bone marrow cells was unaffected. Thus, the in vitro situation does not mimic the in vivo one. Concerning the nonspecific resistance. TBTO reduced macrophage function as shown by impaired splenic clearance of Listeria monocytogenes bacteria. From in vitro studies it is concluded that impaired in vivo splenic clearance was due to a reduction in both the number of adherent cells in the spleen and bacterial digestion on a cell for cell basis. The effect of TBTO on natural cell-mediated cytotoxicity was investigated in a 51Cr-release assay with YAC lymphoma target cells. In this test, the activity of natural killer (NK) cells in the spleen was significantly suppressed in the 80 mg/kg group while NK cell activity of peritoneal cells was unaltered. However, the activity of adherent peritoneal cells (cytotoxic macrophages) was significantly reduced in the 20 and 80 mg/kg groups. Finally, TBTO did not render rats more susceptible to the lethal effects of endotoxin. From the results it is concluded that low-dose feeding of TBTO suppresses thymus-dependent immune responses as well as parameters of the nonspecific resistance.

Comparative toxicologic study with polychlorinated biphenyls in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues

Abstract In a comparative 60-day oral toxicity test (400 ppm) in chickens, three 60%-chlorinated commercial polychlorinated biphenyl (PCB) preparations were used: compound I (Phenoclor DP6), compound II (Clophen A60), and compound III (Aroclor 1260). Using mortality, mean survival time, mean weight, and pathological observations (hydropericardium, abdominal, and subcutaneous edema and centrolobular liver necrosis) as parameters, a significant difference in toxicity was found between the compounds: compounds I and II showed the highest, compound III the lowest, toxicity. Microscopically centrolobular liver necrosis was found in chicks fed compounds I and II. Atrophy of the spleen was found in all test groups. Chemical porphyria was found as a general PCB effect: increased fecal excretion of coproporphyrin and protoporphyrin and fluorescence of tissues occurred in all test groups. Additional experiments with compound I (2000 ppm) in Japanese quail and rats confirmed the porphyrogenic action. Gas chromatographic analyses of liver and brain of dead chicks gave PCB levels that varied between 120 and 2900 ppm. The relationship of hydropericardium (chick edema) and liver necrosis to the differences in toxicity observed between the technical PCB mixtures is discussed.

Toxicity of organotin compounds. II. Comparative in vivo and in vitro studies with various organotin and organolead compounds in different animal species with special emphasis on lymphocyte cytotoxicity.

A series of organotin and organolead compounds were fed to male and female weanling rats for 2 weeks at dietary levels of 0, 50, or 150 ppm, in order to evaluate their toxic effects, with special emphasis on the thymus and peripheral lymphoid organs. A dose-related reduction in the weight of thymus, spleen, and popliteal lymph node, associated with lymphocyte depletion in the cortex of the thymus and the thymus-dependent areas in the peripheral lymphoid organs, was observed in animals fed di-n-octyltin dichloride (DOTC) and di-n-butyltin dichloride (DBTC). As shown previously, these effects were not related to stress. Similar, but less pronounced, lymphoid organ changes occurred in animals fed diets containing diethyltindichloride (DETC) and di-n-propyltindichloride. In contrast, other dialkyltin compounds (dimethyltindichloride; di-n-dodecyltindibromide; and di-n-octadecyltindibromide), as well as mono-n-octyltintrichloride (MOTC), tri-n-octyltinchloride, and tetraoctyltin did not cause atrophy of lymphoid organs. Of the dialkyllead compounds tested, di-n-butylleaddiacetate and di-n-hyxylleaddiacetate, the latter caused distinct lymphoid atrophy, but only when associated with severe growth retardation. A similar structure-activity relationship regarding thymus atrophy by dialkyltin compounds was observed after iv application. DOTC and DBTC were effective at a dose of 1 mg/kg. A dose-related reduction of thymus weight, cell count, and cell viability occurred in rats after a single iv injection of 0, 1, 2, 4, or 8 mg of DOTC/kg. The effect of DOTC was completely reversible. In contrast to rats, lymphoid atrophy did not occur in mice, guinea pigs, or Japanese quail fed DOTC or DBTC. In vitro incubation of thymocytes and bone marrow cells with DBTC and DOTC revealed the same selectivity regarding cell type and species origin of cells as was found in vivo. A dose-related decrease in the survival of rat thymocytes was observed, whereas rat bone marrow cells were not affected by DBTC and DOTC at levels up to 50 μg/ml. In contrast, the number and viability of mouse and guinea pig thymocytes exposed to DBTC were the same as the controls. As the survival of human thymocytes was markedly decreased by DBTC, it possibly acts in the same manner in man and rat. In agreement with in vivo results, the survival of rat thymocytes was not significantly decreased by dimethyltindichloride and diethyltindichloride.

Toxicity of organotin compounds. III. Suppression of thymus-dependent immunity in rats by di-n-butyltindichloride and di-n-octyltindichloride

To evaluate the functional significance of di-n-butyl (DBTC)- and di-n-octyltin dichloride (DOTC)-induced lymphoid depletion various immune function studies were carried out. The delayed type hypersensitivity reaction, a parameter of cell-mediated immunity was decreased in rats fed 50 or 150 ppm of DOTC for 6 weeks. This decrease was dose-related. Allograft rejection, another cellular immune response, was significantly delayed by DBTC and DOTC. The antibody response against E. coli LPS, probably a T cell-independent antigen, was not affected by DBTC. However, the humoral immune response against sheep red blood cells (SRBC), which needs the co-operation of T helper cells and B cells, was distinctly depressed by DBTC. Hemagglutination and hemolysin titers as well as the number of direct plaque-forming cells against SRBC per spleen were decreased in a dose-related manner by DBTC. The phagocytic capacity of macrophages of rats was not affected by DOTC as was shown in the carbon clearance test. Altered immune functions were never found in mice or guinea pigs exposed to DBTC or DOTC. From this study it is concluded that both DBTC and DOTC induce immune suppression in rats by a selective inhibition of T-lymphocyte activity. Immune suppression was most pronounced in animals exposed to the chemicals during the developmental phase of the lymphoid system.

Dermal toxicity studies of technical polychlorinated biphenyls and fractions thereof in rabbits

Abstract A significant difference in toxicity between 3 polychlorinated biphenyl (PCB) preparations was found in a prior study: Clophen A 60 and Phenoclor DP6 showing the highest, Aroclor 1260 the lowest, toxicity (Vos and Koeman, 1970) . A subsequent study revealed the presence of tetra- and pentachlorodibenzofuran in Phenoclor and Clophen (Vos et al. , 1970) . In the present study, application of 118 mg of the 3 PCBs (5 times per wk, for 38 days) on the back skin of rabbits also resulted in differences in toxicity. PCB-induced skin lesions were hyperplasia and hyperkeratosis of the epidermal and follicular epithelium. Histopathology of the liver included centrolobular degeneration, centrolobular liver cell atrophy, focal necrosis, and cytoplasmic hyalin degeneration. Definite hyperplasia and hyperkeratosis of the follicular epithelium of the ear skin were seen after the topical application of fractions of Phenoclor and Clophen eluted from chromatographic columns with 25% diethylether in hexane. The fraction from Aroclor caused a minimal hyperplasia and hyperkeratosis of the follicular epithelium. PCB-induced kidney lesions were hydropic degeneration of the convoluted tubules and tubular dilatation with the presence of casts. This dilatation was demonstrated also by a significantly increased relative percentage of the diameter which corresponds to the space of Bowman. Moreover, thymus atrophy and lymphopenia were found. Fecal coproporphyrin and protoporphyrin excretion was increased.

Antagonistic activity of Lactobacillus casei strain Shirota against gastrointestinal Listeria monocytogenes infection in rats

In the present study, the effect of ingested viable Lactobacillus casei Shirota strain YIT9029 on oral infection with the enteric pathogen Listeria monocytogenes in Wistar rats was investigated. Rats were orally infected with 10(9) viable L. monocytogenes. Starting 3 days before the infection, rats received a daily dosage of 10(9) viable L. casei. It was shown that supplementation of L. casei significantly reduced the numbers of L. monocytogenes in stomach, caecum, faeces, spleen and liver, 2 days after L. monocytogenes infection. The number of L. monocytogenes in the mesenteric lymph nodes was not affected by the ingestion of L. casei. In comparison with control animals, the levels of the liver-specific alanine aminotransferase were lower in L. casei-fed rats. Histological analysis of spleen and liver revealed no differences between the experimental and control animals. In a parallel study with orally L. monocytogenes infected rats, it was shown that L. casei was able to increase cellular immunity significantly as determined with the delayed-type hypersensitivity response against heat-killed L. monocytogenes. In conclusion, in the present study it was shown that orally administered L. casei is able to enhance host resistance against oral L. monocytogenes infection. In the gastrointestinal tract, as well as in the spleen and liver, L. monocytogenes numbers were reduced. Furthermore, it is concluded that the enhancement of this anti-Listeria activity might be, at least partly, due to increased cell-mediated immunity.

Morbillivirus in monk seal mass mortality

We have identified a morbillivirus in organs of Mediterranean monk seals (Monachus monachus), lost during a recent mass die-off. About half of the population of this highly endangered species, which inhabits the Mauritanian coast of West Africa (Cap Blanc), were killed. The outbreak is reminiscent of several recent morbillivirus outbreaks in aquatic mammals.