Analysis of breast milk to assess exposure to chlorinated contaminants in Kazakhstan: sources of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposures in an agricultural region of southern Kazakhstan.

High levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; up to 208 pg/g fat) were measured in samples of breast milk collected in 1997 from 64 donors [41 first-time mothers (primiparae)] living on state farms in southern Kazakhstan. TCDD was the major contributor (70%) to the toxic equivalents, matching the congener patterns found in breast milk and serum samples collected in 1994 and 1996 from donors in nearby villages. The highest TCDD levels were found in state farms adjacent to a reservoir (zone A), which receives agricultural runoff from cotton fields. TCDD levels in zone A were significantly higher than levels in a region more distant (zone B; > 10 miles) from the reservoir (zone A: mean 53 pg/g, n = 17; zone B: mean 21 pg/g, n = 24; p = 0.0017). Levels of TCDD in breast milk and animal-derived foodstuffs were 10 times U.S. levels. Body burden and dietary data suggest that exposures to TCDD are chronic, environmental, and long term and may be related to the use of chemicals in cotton agriculture. The data suggest that the most likely source is the use of cotton defoliants contaminated with TCDD, and the most likely pathway for human exposure is via the consumption of contaminated foodstuffs.

Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) results in a broad spectrum of biological responses, including altered metabolism, disruption of normal hormone signaling pathways, reproductive and developmental effects, and cancer. Cytochrome P450 1B1 (CYP1B1) is a dioxin-inducible gene that is active in the formation of 4-hydroxyestradiol, a potentially genotoxic catechol estrogen. Therefore, the analysis of CYP1B1 in humans may be useful in establishing relationships between dioxin exposure and adverse health effects. In this study, we examined the expression of CYP1B1 in human peripheral blood lymphocytes of unexposed individuals using a quantitative reverse transcription-PCR method. Absolute CYP1B1 RNA levels varied more than 30-fold in uncultured mononuclear cells obtained from 10 individuals. In vitro treatment of mitogen-stimulated lymphocytes with TCDD for 1-5 days of culture resulted in a peak induction of CYP1B1 after 3 days. The induction of CYP1B1 RNA levels after 3 days of culture was dose-dependent, exhibited a maximum response above 10 nM TCDD, and varied greatly among different individuals. However, the half maximal dose required for this induction was similar between individuals and comparable to that observed in the MCF-7 and HepG2 human cell lines. These observations indicate that CYP1B1 exhibits variable constitutive expression and is inducible in vitro by TCDD in human lymphocytes and that the magnitude of induction varies within the population. These data define the suitability of CYP1B1 for use as a mechanistically based biomarker in ongoing molecular epidemiological studies of human populations exposed to dioxins and related chemicals that bind the aromatic hydrocarbon receptor.

Analysis of breast milk to assess exposure to chlorinated contaminants in Kazakstan: high levels of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in agricultural villages of southern Kazakstan.

To assess levels of chlorinated contaminants in breast milk, we measured organochlorine pesticides, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) in breast milk samples collected in 1994 according to the World Health Organization protocol from 92 donors that were representative of regional populations in southern Kazakstan. High levels (10-120 pg/g fat) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic of the PCDD/PCDF congeners, were found in breast milk samples from an agricultural region. TCDD was the major contributor (75%) to the international toxicity equivalents of these samples. The same distinctive PCDD/PCDF congener pattern was found in 15 breast milk samples and 4 serum samples collected in 1996 in a follow-up study, and has now been confirmed by three analytical laboratories.

Animal models of human response to dioxins.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent member of a class of chlorinated hydrocarbons that interact with the aryl hydrocarbon receptor (AhR). TCDD and dioxinlike compounds are environmentally and biologically stable and as a result, human exposure is chronic and widespread. Studies of highly exposed human populations show that dioxins produce developmental effects, chloracne, and an increase in all cancers and suggest that they may also alter immune and endocrine function. In contrast, the health effects of low-level environmental exposure have not been established. Experimental animal models can enhance the understanding of the effects of low-level dioxin exposure, particularly when there is evidence that humans respond similarly to the animal models. Although there are species differences in pharmacokinetics, experimental animal models demonstrate AhR-dependent health effects that are similar to those found in exposed human populations. Comparisons of biochemical changes show that humans and animal models have similar degrees of sensitivity to dioxin-induced effects. The information gained from animal models is important for developing mechanistic models of dioxin toxicity and critical for assessing the risks to human populations under different circumstances of exposure.

Acquired risk factors and susceptibility to environmental toxicants.

Susceptibility to illness after exposure to environmental toxicants is determined by the interaction of numerous factors involving both constitutive and acquired traits. Constitutive susceptibility (risk) factors are the intrinsic traits determined by developmental stage, gender, and genetic makeup. Within a population, changes in constitutive risk factors tend to occur slowly, through aging, alterations in the birth or death rate, or by migration in or out of the population. Often overlooked is the effect of acquired susceptibility factors on susceptibility to environmental toxicants. Acquired susceptibility factors, which are related to the effects of living conditions, psychosocial factors, diet, behavior and access to medical care, may modify the effect of constitutive factors. Three examples demonstrate the interaction of acquired susceptibility factors with exposure and constitutive factors. The increased prevalence of asthma in children is suspected of having a strong environmental component but the underlying acquired susceptibility factors, if any, are difficult to identify because of the multifactorial nature of asthma and the use of surrogate risk factors such as parent's education. ß-Carotene is a dietary component which may modify acquired susceptibility. While numerous observational studies find that dietary ß-carotene reduces the risk of lung cancer in cigarette smokers, intervention studies do not support this role. Hepatitis B is an example of an infectious agent functioning as an acquired susceptibility factor. Hepatitis B synergistically increases the risk of hepatocellular carcinoma when accompanied by exposure to aflatoxin, a relationship that may be modified by constitutive risk factors, such as epoxide hydrolase capabilities. Acquired risk factors have the potential to greatly influence risk and their impact should be included in future studies of the health effects of environmental toxicants.

Obtaining information about susceptibility from the epidemiological literature.

Whether people become ill after encountering environmental pollutants depends on the magnitude of their exposure and their capacity to respond. Exposure and intrinsic response capabilities vary within the population. Those that become ill when the general population remains largely unaffected are considered to be highly susceptible. The U.S. Environmental Protection Agency (USEPA), responsible for protecting the public from environmental pollutants, has developed risk assessment procedures to assist in evaluating the likelihood of health effects. However, the Agency's ability to evaluate the risk faced by highly susceptible populations is often hindered by the paucity of adequate health effects data. Response variability can be assessed with animal models and human epidemiological studies. Although animal models are useful when evaluating the effect of gender and developmental stage on susceptibility, inbred rodent strains underestimate the genetic and lifestyle-induced variability in susceptibility found in human populations. Epidemiological approaches are the preferred source of information on variability. This paper reviews the epidemiological literature from the perspective of a risk assessor seeking data suitable for estimating the risk to highly susceptible populations. Epidemiological approaches do not measure the full range of population response variability. Rather, "susceptibility factors" are evaluated either as risk factors or by focusing on the susceptible population, e.g. children. Susceptibility factors due to genetics, developmental stage, gender, ethnicity, disease state and lifestyle are most frequently encountered. Often, the information describing the health impact of the susceptibility factor is incomplete due to, (1) a failure to consider factors modifying susceptibility; (2) inadequate exposure data; (3) a failure to evaluate the health impact of the susceptibility factor. In addition, for a given exposure agent, several susceptibility factors may be relevant. While incomplete data describing susceptibility factors limits the opportunity for quantitative estimations of risk, available information can supplement qualitative evaluations and risk management.

Development of an immunoassay to detect hemoglobin adducts formed by benzene exposure.

Polyclonal murine antibodies that recognize the adducts formed by benzene metabolites in hemoglobin (Hb) were prepared and used to develop immunoassays. In competitive inhibition assays, the concentration of competitor needed to reduce the signal by 50% (IC50) was less than 10 pmoles for hydroquinone-hemoglobin (HQHb) adducts and less than 1 pmole for 1,2,4 trihydroxybenzene-hemoglobin (TriOH Hb). Hemoglobin (Hb) incubated with either phenol or catechol (CAT) did not elicit antibodies suitable for quantitative immunoassays. The metabolite-directed immunoassays were tested using hemoglobin from mice previously administered [C14] benzene for two to four weeks. The most sensitive assay for hydroquinone measured 0.49 pmoles adduct/40 pmoles Hb (191 pmoles adduct/mg Hb) in mice treated with 200 mg/kg benzene (P < 0.05, Student's t test). TriOH Hb adducts were not detected.

Immunochemical techniques in biological monitoring.

Immunoassays are analytical methods that detect interactions between antibodies and antigens. Immunoassays were used originally to detect large biological molecules. The new generation of these antibody-based assays can detect small synthetic compounds. As a result, immunoassays are being developed specifically for biomarkers of exposure and effect to environmentally prevalent chemicals. Immunochemical detection of parent compounds in blood and tissues, metabolites in excreta, and adducts with DNA and protein have been successfully performed by several investigators. Although there is great potential for use of immunoassays in biological monitoring studies, the limitations of these analyses must be fully understood to prevent improper evaluation of the acquired data. This review will cover some of the background material necessary to understand how an antibody-based assay is developed. The differences between polyclonal and monoclonal antibody-based assays and the importance of antibody class, affinity, specificity, and cross-reactivity must be considered in both study design and data analysis.