Integrating epidemiology and toxicology in neurotoxicity risk assessment

Neurotoxicity risk assessments depend on the best available scientific information, including data from animal toxicity studies, human experimental studies and human epidemiology studies. There are several factors to consider when evaluating the comparability of data from studies. Regarding the epidemiology literature, issues include choice of study design, use of appropriate controls, methods of exposure assessment, subjective or objective evaluation of neurological status, and assessment and statistical control of potential confounding factors, including co-exposure to other agents. Animal experiments must be evaluated regarding factors such as dose level and duration, procedures used to assess neurological or behavioural status, and appropriateness of inference from the animal model to human neurotoxicity. Major factors that may explain apparent differences between animal and human studies include: animal neurological status may be evaluated with different procedures than those used in humans; animal studies may involve shorter exposure durations and higher dose levels; and most animal studies evaluate a single substance whereas humans typically are exposed to multiple agents. The comparability of measured outcomes in animals and humans may be improved by considering functional domains rather than individual test measures. The application of predictive models, weight of evidence considerations and meta-analysis can help evaluate the consistency of outcomes across studies. An appropriate blend of scientific information from toxicology and epidemiology studies is necessary to evaluate potential human risks of exposure to neurotoxic substances.     

An in vitro approach to assess the toxicity of certain food contaminants: methylmercury and polychlorinated biphenyls

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants and food contaminants, and known developmental neurotoxicants. Aim of this study was to evaluate the effects of MeHg, PCB 126 and PCB 153 in a battery of in vitro cell systems. A total of 17 cell types were utilized, including nervous system (neuronal and astroglial) and non-nervous system cells. End-points measured included MTT reduction, Trypan blue exclusion and (3)H-thymidine incorporation into DNA. Results indicate that this approach would identify these three compounds as neurotoxicants, and would also point out to the thyroid (for PCB 126 and MeHg) and the prostate (for both PCBs) as important additional targets. Tests of binary combinations of MeHg and PCBs indicated no interaction and an additive response, in agreement with other recent reports. Cerebellar granule neurons from mice with genetically determined low glutathione levels were more sensitive than wild-type neurons to the toxicity of all three compounds, supporting a role for oxidative stress in their neurotoxicity. These findings provide initial evidence that a relatively rapid in vitro screening approach can be developed, that would provide initial information useful for assessing neurotoxicity, as well as indication on potential other targets of biological action or toxicity.     

Convergence of clinical toxicology and epidemiology in relation to health effects of chemicals

An integrated approach of clinical toxicology and epidemiology is an essential ingredient in environmental health risk management through molecular epidemiology and environmental genomics. The last decade has also seen the emergence of several biochemical markers useful in chemical risk assessment and in epidemiological studies. An appraisal of the concepts involved, the approaches required, and the potential scope of this approach is attempted here.     

Biological responses of the rat to polychlorinated biphenyls

A commercial polychlorinated biphenyl mixture (PCBs), Aroclor 1242, was administered to rats po by intubation in order to determine toxic manifestations of acute and subacute ingestion. In addition, the effect of PCBs on hepatic microsomal enzyme systems in rats was evaluated. The oral, 14-day LD50 was determined to be approximately 4.25 g/kg. Major toxic signs observed upon administration of high doses of PCBs included diarrhea, chromodacryorrhea, loss of body weight, unusual stance and gait, lack of response to pain stimuli, and terminal ataxia. Progressive dehydration and CNS depression appeared to be contributing factors in each fatality. Histopathologic alterations were evident only in the liver and kidneys, manifest as foci of sudanophilic vacuolation. Rats maintained on an oral dosage regimen of 100 mg/kg every other day for 3 weeks exhibited similar histopathologic changes, but no overt signs of toxicity. Serum GOT activities were elevated over controls in both the acute and subacute groups. A single ip injection (100 mg/kg) increased liver weight, total hepatic microsomal enzyme activity (measured as hydroxylation of acetanilide and N-demethylation of aminopyrine), and hepatic cytochrome P₄₅₀ and b₅ levels. Hepatic microsomal enzyme activity remained elevated 10 days after a single dose of PCBs, suggesting that PCBs may play an important role in altering biologic responses of mammals subjected to environmental chemical stress.     

Serum levels of polychlorinated biphenyls in Mexican women and breast cancer risk

The most prevalent female cancer across the world is breast cancer. Current established breast cancer risk factors explain only a fraction of the breast cancer cases diagnosed, and for this reason, other environmental factors have been studied. Exposure to organochlorine compounds has been linked to an increased incidence of breast cancer, although not all data have been consistent. This study was designed to evaluate the relation between polychlorinated biphenyls (PCB) exposure and breast cancer risk in Mexican women. We recruited 140 women from the General Hospital. The cases were 70 newly diagnosed women. We collected environmental and reproductive information by questionnaire. Blood samples were taken for measurement of serum levels of 20 PCB congeners. Risk of breast cancer was found to be positively associated with heavy congeners, age, postmenopausal status, family history of breast cancer and living close to an industrial facility. When PCB were grouped by structure–activity relationships, the risk of breast cancer was positively associated with groups 2b (odds ratio, OR = 1.90, 95% confidence interval, CI, 1.25–2.88), 3 (OR = 1.81, 95% CI 1.08–3.04) and group 4 (OR = 1.57, 95% CI 1.20–2.07). Among postmenopausal women, PCB levels from groups 1a, 2b, and 4 and total PCB were higher in cases, and an association between risk of breast cancer with groups 1a (OR = 7.59, 95% CI 1.1–51.4), 2b (OR = 3.7, 95% CI 1.2–11.2) and 4 (OR = 1.8, 95% CI 1.1–3.1) was found in this group of women. This study showed an association between heavy and potentially estrogenic PCB congeners and breast cancer risk. Copyright © 2011 John Wiley & Sons, Ltd.     

The use of epidemiology and clinical toxicology to assess environmental health problems

Historically, epidemiology has played an important role in the changes that have taken place over time as epidemics of important infectious diseases have been replaced with modern epidemics of chronic, degenerative diseases such as elevated blood pressure, various types of cancers, diabetes, and stroke, among others. Two illustrations of the early uses of epidemiologic methods in investigations of disease outbreaks are described in the work of John Snow, who described outbreaks of cholera in the 1800s (1936, Snow on Cholera, Commonwealth Fund, New York), and J. Goldberger, who investigated the incidence of pellagra in the early 1900s (1964, Goldberger on Pellagra, M. Terris, ed., Louisiana State Univ. Press, Baton Rouge). The more contemporary use of epidemiology and clinical toxicology to assess the outcome of human exposure to environmental contaminants in the food supply are described for exposure to the polychlorinated biphenyls (PCBs) and to the polybrominated biphenyls (PBBs). Exposure to the PCBs has occurred in a variety of locations worldwide, with the greatest exposure taking place in individuals of many countries who consume fish and in Japan and Taiwan through contaminated cooking oil. Exposure to PBBs has essentially been limited to the State of Michigan where widespread contamination of cattle, dairy products, and poultry has taken place.     

Exposure to polychlorinated biphenyls and hearing impairment in children

The objective of this cross-sectional epidemiological study was to assess if long-term exposure to polychlorinated biphenyls (PCBs) is associated with hearing impairment. Four hundred and thirty-three children aged 8-9 years residing in an area polluted by PCBs in Eastern Slovakia were examined otoscopically, tympanometrically and by pure tone audiometry. PCB levels in their serum were determined by gas chromatography. Transient otoacoustic emissions (TEOAE) were measured in a subgroup of 161 children. The mean of the sum of PCB concentrations in serum was 528.2ng/g serum lipids (median 321ng/g serum lipids). Serum PCB concentrations were associated with an increase of hearing threshold at low frequencies and a negative correlation between serum PCBs and the amplitude of TEOAE response was observed in the uppermost tertile of children grouped with regard to serum PCBs, not related to thyroid hormone levels. It was concluded that long-term environmental exposure to PCBs is associated with subclinical but diagnosable hearing deficits.     

Determination of selected organochlorine pesticides and polychlorinated biphenyls in human serum.

A method is presented that can be used to determine the residue level of certain chlorinated pesticides and polychlorinated biphenyls (as Aroclor 1260) in serum. The method involves the following: (1) extraction of denatured serum with organic solvents; (2) elution of the organic extract through micro-Florisil columns to obtain two fractions; (3) acid treatment of the less polar Florisil fraction and its subsequent elution through deactivated silica gel to obtain two fractions; and (4) analysis of all three fractions using gas-liquid chromatography with electron capture detection. The method produced in vitro recoveries for 10 pesticides spiked in the range of 1-10.7 ppb of 50.4% to 121.6%, and in the range of 4.98-21 ppb, recoveries ranged from 47.7 to 112.6. In vivo "recoveries" of Aroclor 1260 averaged 104.8% and 92.3% for concentration levels of approximately 10 and approximately 30 ppb, respectively. The method could not be compared with the more commonly used hexane extraction technique because of the deleterious effect these extracts had on the gas chromatographic system.     

A review of the Food and Drug Administration risk analysis for polychlorinated biphenyls in fish

This paper reviews the Food and Drug Administration (FDA) analysis of the risk to humans from consuming fish contaminated with polychlorinated biphenyls (PCBs). In brief, the FDA methodology employed "high" dose experiments on animals and extrapolated the observed rates of certain types of cancer at these elevated doses to the low doses found in human diets. These extrapolations were then used to define a recommended tolerance level of 5 ppm, and proposed reduction to 2 ppm, for fish sold in interstate commerce. Unfortunately, as is shown here, such a procedure is extremely sensitive to the basis for extrapolation. Important elements of the FDA analysis include the following: (i) FDA assumed a particular form of the dose-response model: the one-hit model. Many other models have been proposed and, on balance, appear equally plausible. These models estimate lower risks than does the one-hit model. (ii) FDA calculated 99% upper confidence bounds on these risks and, moreover, emphasized cases of fish eaters who consume greater amounts of PCB-contaminated fish than do 98.5% of the U.S. population. (iii) FDA based PCB ingestion computations on consumption of raw fish, whereas most fish are cooked before eating, and it is known that PCB levels in cooked fish are lower than PCB levels in raw fish. (iv) FDA based estimates of cancer risk on the assumption that PCB levels in fish would be constant over the nominal 70-year human life span used in the FDA "lifetime risk" computation. Recent data suggest that PCB levels have been declining in fish (particularly in sport fish) and humans as well. Such trends imply significantly lower cumulative lifetime PCB doses than were assumed in the FDA analysis. (v) FDA assumed that humans and test animals are equally sensitive to PCB ingestion when measured on a parts per million in diet basis. Extrapolations on an equivalent consumption per unit of body weight, thought appropriate by most researchers, result in much lower health risks. In short, when confronted with methodological choices, the FDA consistently selected "worst case" or conservative assumptions over other alternatives of at least equal plausibility. This philosophy of choice was explicitly acknowledged by the FDA. What was omitted from the FDA analysis, however, was the possible degree of overstatement of these risks. The results of replicate risk computations using alternative assumptions to examine the possible magnitude of overstatement of health risk are summarized in Table 12. As can be seen, this overstatement could easily account for a discrepancy of several orders of magnitude between actual and calculated risks.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of epidemiology in the regulation of dioxins in the food supply

Residues of tetrachlorodibenzo-p-dioxins (TCDD) were detected recently in several species of freshwater fish from various areas of the Great Lakes. Concern for the potential human health risk associated with the consumption of these fish prompted the Food and Drug Administration to assess epidemiological and toxicological TCDD data in order to determine the need for regulatory action. In the assessment, pertinent animal and human data and data for the TCDD residues in the freshwater fish, for the amounts of the different fish species consumed and for the number of individuals sho consumed the fish, were evaluated. The various methods of evaluation used to reach a regulatory decision are described.     

Prenatal exposure to polychlorinated biphenyls and dioxins is associated with increased risk of wheeze and infections in infants

The birth cohort BraMat (n = 205; a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health) was established to study whether prenatal exposure to toxicants from the maternal diet affects immunological health outcomes in children. We here report on the environmental pollutants polychlorinated biphenyls (PCBs) and dioxins, as well as acrylamide generated in food during heat treatment. The frequency of common infections, eczema or itchiness, and periods of more than 10 days of dry cough, chest tightness or wheeze (called wheeze) in the children during the first year of life was assessed by questionnaire data (n = 195). Prenatal dietary exposure to the toxicants was estimated using a validated food frequency questionnaire from MoBa. Prenatal exposure to PCBs and dioxins was found to be associated with increased risk of wheeze and exanthema subitum, and also with increased frequency of upper respiratory tract infections. We found no associations between prenatal exposure to acrylamide and the health outcomes investigated. Our results suggest that prenatal dietary exposure to dioxins and PCBs may increase the risk of wheeze and infectious diseases during the first year of life.     

Serum hormones in boys prenatally exposed to polychlorinated biphenyls and dibenzofurans

Polychlorinated biphenyls (PCBs) and dibenzofurans (PCDFs) are persistent environmental pollutants shown to adversely interact with several functions of the endocrine system. In 1978-1979, over 2000 Taiwanese people ingested rice oil accidentally contaminated with PCBs and PCDFs. This is one of the major toxic exposure episodes that occurred globally and was later called Yucheng (oil disease in Chinese). The children born to exposed Yucheng women were therefore exposed in utero to high doses of PCBs/PCDFs. In 1995, 60 Yucheng and 61 control boys participated in physical examination, and serum hormones were measured by radioimmunoassay (RIA). Age, body weight, body height, Tanner status, testicular size, serum luteinizing hormone (LH), prolactin (PRL), thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) levels were not statistically different between Yucheng and control boys in the subgroups of before and at the age of puberty. However, the serum estradiol (E2) levels were significant higher in Yucheng boys at the age of puberty. Yucheng and control boys were further divided into two subgroups, those before (age <13 yr) and those at the age of puberty (age > or = 13 yr). There was a decrease of serum testosterone (TT) levels and increase of serum follicle-stimulating hormone (FSH) levels in Yucheng boys at the age of puberty as compared with controls. There was a significant decrease of the square root of TT/E2 and TT/FSH; however, the square root of E2/FSH was increased in Yucheng boys at the age of puberty as compared with controls. Data indicated that prenatal exposure to PCBs and PCDFs may have implications for boys' sex hormone homeostasis at puberty. Further studies are needed to identify the congeners of PCBs/PCDFs responsible for disruption of the endocrine system, as well as the mechanisms of such disruption.     

Assessment of the Serbian population exposure to polychlorinated biphenyls by crops

Data on occurrence of six marker polychlorinated biphenyls (PCBs) in 35 composite samples of some crop products (wholegrain wheat flour, edible sunflower oil and white sugar) and by-products of food processing industry (bran, dried sugar beet pulp and molasses) were collected and combined with food consumption data to assess the dietary intake of these persistent food contaminants by the Serbian adult population. The average daily intake of sum of PCBs was assessed to be 172.2ng using a mean weight of 60kg for the general population in Serbia. The wheat-based products contributed largely (141.0ngday(-1)) to the estimated value, due to fact that these products are notably consumed in Serbia, while the contribution of edible oil (19.8ngday(-1)) and sugar (11.4ngday(-1)) were not pronounced. The estimated daily intakes were compared with the maximum permissible risk (MPR) level of 600ngday(-1) established by the Dutch National Institute for Public Health and the Environment. The additional source seemed to be found in the market basket that included meat products, fish, dairy products and eggs.     

Developmental and neurobehavioral effects of perinatal exposure to polychlorinated biphenyls in mice

Because behavioral deficits associated with gestational exposure to polychlorinated biphenyls (PCBs) have been a concern, we studied the developmental and neurobehavioral effects of perinatal exposure to Aroclor 1254 (A1254), a commercial mixture of PCBs, in mice. The PCB mixture (A1254; 0, 6, 18, and 54 mg/kg body weight) was administered to pregnant mice (C57BL/6Cr) every 3 days by gavage from gestational day (GD) 6 to postnatal day (PND) 20. Compared with the control, treatment with A1254 did not alter the maternal body weight during the gestation and lactation periods. The body weight of the offspring did not differ among treatments. To assess the effects on offspring following such exposure, physical and neurobehavioral development (i.e., pinna detachment, hair growth, eye opening, incisor eruption, grasp reflex, righting reflex, walking, negative geotaxis, and cliff avoidance) was observed before weaning. At PND 7, poor adult-like responses in negative geotaxis were observed in all exposed groups. When the offspring were at 8-week old, the PCB-treated (18 mg/kg body weight) mice showed a decreased walking speed in the open-field test, and a prolonged time to reach the platform in the water maze test. Spontaneous locomotion activity was not affected by PCB exposure at 9 weeks . These results showed that perinatal exposure to PCBs produces several behavioral alterations in mice. Although dose-dependent changes were not observed, the neurobehavioral effects such as a decreased walking speed in the open-field test and a prolonged time to reach the platform in the water maze test remained in adulthood after the seeming recovery from the transient delay in development before weaning.     

Distribution and biliary excretion of polychlorinated biphenyls in rats

Absorption, distribution, and excretion of two ³H-labeled polychlorinated biphenyls (PCB), 2,4,5,2′,4′,5′,-hexachlorobiphenyl (HCB) and 2,5,2′,5′-tetrachlorobiphenyl (TCB), were studied in surgically prepared male and female rats. Approximately 3–5 hr after surgery, HCB or TCB (50 mg/kg) was administered into the stomach. Bile, urine, and feces were collected for 24 hr after which the animals were sacrificed and tissues taken for determination of ³H content. The distribution of ³H remaining in the rats, expressed as percentage of dose, was highest in skeletal muscle, skin, liver, and small intestine for both isomers. The major difference observed between the PCBs was in biliary excretion. For HCB, 0.5 ± 0.2% (males, mean ± SE) and 1.1 ± 0.3% (females) of the dose were excreted in bile in 24 hr; for TCB, 42.2 ± 8.5% (males) and 25.7 ± 7.8% (females) were excreted by the same route. The lower biliary excretion of HCB than of TCB cannot be accounted for by a difference in absorption from the gastrointestinal tract and is thought to be due to a slower rate of HCB metabolism. More explicitly, the chlorines in the 4,4′ positions of HCB appear to prevent rapid biliary excretion of the compound by eliminating adjacent unsubstituted carbons which are necessary for rapid metabolism to occur. Urinary excretion of HCB and TCB was of minor importance compared to biliary excretion. Generally, absorption, distribution, and excretion of the PCBs were similar in males and females.     

Augmented atherogenesis in ApoE-null mice co-exposed to polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) are persistent organic pollutants found as complex mixtures in the environment throughout the world. Therefore, humans are ubiquitously and simultaneously exposed to TCDD and PCBs. TCDD and PCBs alone have been linked to atherosclerosis. However, the effects of interactions or synergism between TCDD and PCBs on atherogenesis are unknown. We investigated the possible enhanced atherogenesis by co-exposure to TCDD and PCBs and the potential mechanism(s) involved in this enhancement. Male ApoE^−/− mice were exposed to TCDD (15 μg/kg) and Aroclor1254 (55 mg/kg, a representative mixture of PCBs) alone or in combination by intraperitoneal injection four times over six weeks of duration. Our results showed that mice exposed to TCDD alone, but not Aroclor1254 alone, developed atherosclerotic lesions. Moreover, we found that atherosclerotic disease was exacerbated to the greatest extent in mice co-exposed to TCDD and Aroclor1254. The enhanced lesions correlated with several pro-atherogenic changes, including a marked increase in the accumulation of the platelet-derived chemokine PF4, and the expression of the proinflammatory cytokine MCP-1 and the critical immunity gene-RIG-I. Our data demonstrated that co-exposure to TCDD and Aroclor1254 markedly enhanced atherogenesis in ApoE^−/− mice. Significantly, our observations suggest that combined exposure to TCDD and PCBs may be a greater cardiovascular health risk than previously anticipated from individual studies.     

Effect of polychlorinated biphenyls and polychlorinated quaterphenyls in Cynomolgus monkey (Macaca fascicularis)

Female Cynomolgus monkeys (Macaca fascicularis) with P-KC-400, Y-PCB, PY-PCB or polychlorinated quaterphenyls (PCQ) received a daily dose of 5 mg for 20 weeks, and some monkeys received a daily dose of 10 mg of Y-PCB or 0.5 mg of PCQ. The chemical compositions of the polychlorobiphenyls (PCB) used for the oral administration were as follows: P-KC-400, PCB from which polychlorodibenzofurans (PCDF) have been removed from Kanecklor 400, largely contains tri- and tetrachlorobiphenyls and no PCDF. Whereas, Y-PCB and PY-PCB, PCB with constituents similar to PCB ingested by yusho patients, largely contain penta- and hexachlorobiphenyls, in addition, PCDF of 400 ppm was present only in Y-PCB, but not in PY-PCB. There were immunosuppression, enlargement and histopathological changes of the liver (such as interstitial inflammation, and proliferation of epithelial cells of biliary duct, etc.) in the groups fed P-KC-400 and PY-PCB (free of PCDF). In the group fed Y-PCB (with PCDF), there were more apparent decreases in body weight, immunosuppression, fatty liver and histopathological changes than in the groups P-KC-400 and PY-PCB. In addition, there were hair loss, acneform eruptions, edema of the eyelid, congestion and abscess of the Meibomian gland, and cornifications of the skin, characteristic dermatological findings of yusho disease.     

Distribution and effect of some polychlorinated biphenyls in the hemopoietic tissues

Distribution of 14C-labeled 2,4',5-trichlorobiphenyl 2,2',4,4',5,5'-hexachlorobiphenyl, and 2,2',3,3',4,4',5,6'-octachlorobiphenyl was studied in the hematopoietic tissues of squirrel monkey and mice (C67 Bl) using whole-body autoradiography. An accumulation of radioactivity was seen in the bone marrow of monkey 24 h and 48 h after iv injection of hexachlorobiphenyl and trichlorobiphenyl, respectively. High concentration of radioactivity was also observed in the bone marrow of normal mice injected iv with octachlorobiphenyl. A combination of whole-body autoradiography and spleen-colony assay in supralethally irradiated mice implanted with syngeneic bone-marrow cells indicated the major part of radioactivity to be localized outside the bone-marrow hemic compartment, probably in the fat. No effect of hexachlorobiphenyl but a significant inhibition with octachlorobiphenyl (10(-5) M) and especially trichlorobiphenyl (10(-5) M) was seen on the formation of granulocytic colonies from mouse progenitor cells in vitro.     

Use of primate folliculogenesis models in understanding human reproductive biology and applicability to toxicology

The nonhuman primate reproductive system provides an excellent model for studying basic physiological processes applicable to humans. This article reviews hormonal observations and experimental manipulations useful in the evaluation of ovarian events in various stages of the reproductive life. As the need arises, primate reproductive toxicological studies may clarify questions relevant to human risk evaluations. Evaluation of reproductive toxicological observations may reveal biological parameters defining premature reproductive failure.