Biomarkers Of Environmental Exposure Research Articles

Association between biomarkers of environmental exposure and increased risk of breast cancer.

* University of Pittsburgh Cancer Institute, Center for Environmental Oncology, 5150 Centre Avenue, Suite 435, Pittsburgh, Pennsylvania, 15232, USA. University of Pittsburgh Graduate School of Public Health. Department of Epidemiology, Department of Environmental and Occupational Health, Pittsburgh, Pennsylvania, USA. International Agency for Research on Cancer and Institut National de la Sante et de la Recherche Medicale, Lyon, France, now with University of Bordeaux, Department of Preventive Medicine, France. Correspondence to D.L.D. e-mail: davisdl@upmc.edu

Aflatoxin B1 and polycyclic aromatic hydrocarbon adducts, p53 mutations and p16 methylation in liver tissue and plasma of hepatocellular carcinoma patients

Elevated aflatoxin B(1)-albumin adducts (AFB(1)-Alb) have been associated with an increased risk for HCC development. However, there are no studies in humans, correlating albumin adducts in blood with liver DNA adducts. Forty frozen tumor tissues and 39 paired plasma samples from HCC patients were collected in Taiwan, to determine the relationship between albumin adducts in blood and DNA adducts in liver tissue as well as mutations in p53 and methylation of p16. AFB(1)- and polycyclic aromatic hydrocarbon (PAH)-DNA adducts in tissue and albumin adducts in plasma were determined by immunohistochemistry and competitive ELISA, respectively. Plasma AFB(1)-Alb adducts in subjects with low, medium and high levels of AFB(1)-DNA adducts in tumor tissues were 51.0 +/- 36.5, 70.5 +/- 48.1 and 84.9 +/- 48.2 fmol/mg, respectively (p(trend) = 0.05). No significant correlation was found for PAH. Fourteen of 40 (36%) tissues were positive for mutant p53 protein by immunohistochemistry; 11 of 40 tissue DNA samples (28%) were positive for p53 mutations, but not their corresponding plasma DNAs. p16 was methylated in 24 of 40 (62%) tissues and 12 of 39 (32%) plasma DNAs. Significant correlations were observed between AFB(1)-Alb adducts and p53 mutations and p16 methylation. These data suggest that genetic, epigenetic and environmental exposure biomarkers in plasma may help in estimating the risk for the development of HCC.

Tail hair as an indicator of environmental exposure of cows to lead and cadmium in different industrial areas

This study examines the use of tail hair from cows as a possible biomarker of environmental exposure to lead and cadmium around different industrial areas. Respective blood and tail hair samples were collected from a total of 317 apparently healthy cows above 3 years of age. This includes 287 cows reared in industrial and urban areas and 30 cows from areas free from polluting sources. Significantly ( P < 0.05 ) higher lead and cadmium residues were recorded in hair from cows reared around lead–zinc smelter and closed lead cum operational zinc smelter. However, cows from those areas had significantly ( P < 0.05 ) higher blood lead but not cadmium concentration as compared to respective control value. Although mean blood lead concentration in cows around aluminum processing plant and urban cum small industrial areas and that of cadmium around steel processing plant were significantly ( P < 0.05 ) higher than respective control, the mean hair lead and cadmium content remained statistically ( P > 0.05 ) comparable to that of respective control values. The blood lead was significantly correlated with hair lead ( r = 0.672 , P < 0.01 ) and cadmium ( r = 0.309 , P < 0.05 ) . There was a significant correlation between lead and cadmium concentration ( r = 0.610 , P < 0.01 ) in hair and a nonsignificant correlation between blood and hair cadmium suggesting that cadmium accumulation in hair was influenced by blood and hair lead concentrations in cows environmentally exposed to lead.

Complementing the Genome with an “Exposome”: The Outstanding Challenge of Environmental Exposure Measurement in Molecular Epidemiology

The sequencing and mapping of the human genome provides a foundation for the elucidation of gene expression and protein function, and the identification of the biochemical pathways implicated in the natural history of chronic diseases, including cancer, diabetes, and vascular and neurodegenerative

Systems Biology: The Big Picture

Genomics, proteomics, and metabolomics have all vastly advanced our understanding of human biology and disease. But the functioning of even a simple system such as a single yeast cell or bacterium is much more complicated than the sum of its genes or proteins or metabolites; it’s the activity of all those components and their relationships to one another that add up to a living organism. Recognizing that complexity, the emerging field of systems biology attempts to harness the power of mathematics, engineering, and computer science to analyze and integrate data from all the “omics” and ultimately create working models of entire biological systems. “Traditionally, scientists—toxicologists included—have relied on a reductionist approach to biology,” says William Suk, director of the NIEHS Center for Risk and Integrated Sciences. Even now, many studies examine complex systems by looking at cellular components in isolation. For instance, a common experiment involves using DNA microarrays to observe the effect of a chemical exposure on thousands of genes at once. This technique can quickly tell a scientist which genes may be vulnerable to that exposure. But a systems biology approach would attempt to model not only the chemical’s effect on gene expression but also how that expression will affect protein function, and in turn how the exposure will affect cell signaling. “There’s nothing wrong with what we’ve been doing,” Suk says. “But systems biology is going to take it to another level.”

Urine Analysis for the Evaluation of Environmental Exposures to Aromatic Hydrocarbons

The authors have developed a dynamic headspace (purge-and-trap) gas chromatographic method, with photoionization detection, for the determination of benzene (C6H6), toluene (C7H8), ethylbenzene (C8H10), and isomeric (o-, m-, p-) xylenes (C8H10) (BTEX) in urine. Detection limits ranged between 15 and 35 ng/l, relative standard deviations between 0.2 and 10%, and accuracy between 80 and 100%. The primary objective of this study was to use this new method to establish baseline concentration data for BTEX in the urine of the general population of Zagreb, Croatia. A second objective was to evaluate the effect of cigarette smoking on those baseline values. BTEX were analyzed in the urine of 72 subjects (36 nonsmokers and 36 smokers) without occupational exposure to BTEX. The nonsmokers had measurable BTEX in their urine, except for ethylbenzene in 13 and o-xylene in 15 of the samples. Values for BTEX were markedly higher among smokers than nonsmokers. Because the sources of BTEX exposure are commonly derived (i.e., vehicle exhausts and smoking), their values in subjects' urine were significantly intercorrelated. Levels of toluene and o-xylene were correlated significantly with the number of cigarettes smoked per day. The use of purge-and-trap gas chromatography with photoionization detection to determine BTEX in urine offers a convenient approach for biological monitoring of the general population. Study data provide referent values for BTEX in urine, which can be used as biomarkers for environmental exposures. Smoking contributes significantly to the urinary concentration of BTEX.

Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style

The original purpose of our study was to determine if the detection of chromosomal aberrations in peripheral lymphocytes of children might be used as a biomarker of environmental pollution and life style. We compared the results of cytogenetic analyses performed in children and adolescents in the periods 1984–1993 and 1994–1999, in a total of 3402 subjects. The frequency of aberrant cells (AB.C.) markedly decreased in the period 1994–1999 compared with the period 1984–1993. The decreases in AB.C. were significant in the age groups 7–15 and 16–19 years: 1.63% AB.C. versus 1.14% AB.C. and 2.02% AB.C. versus 1.08% AB.C., respectively ( P<0.01). No difference in the frequency of AB.C. was observed in newborns. Based on our experience, we believe that monitoring the spontaneous level of chromosomal aberrations in children over 5 year periods may be used to examine the general changes in environmental pollution in larger geographic areas.

Molecular epidemiology and cancer: promising areas for future research in the post-genomic era

This manuscript addresses how epidemiology might benefit from the sequencing of the human genome in terms of identifying the aetiology of cancer. Given that most human cancer is the result of both genetic and environmental risk factors, accurate assessment of both is required to develop an understanding of molecular mechanisms of carcinogenesis. Sequencing the human genome is a major scientific advance, which needs to be considered in the context of the multifactorial aetiology of cancer if it is to bring the maximum benefit. Likewise, assessment of environmental exposure is challenging for a number of reasons, particularly when exposures are relatively low level and vary over time. Biomarkers of environmental exposures (e.g. carcinogen–DNA and –protein adducts) offer the potential to overcome some of these limitations. Furthermore, markers of genetic alterations may permit the detection of relevant early stages of malignancy to inform surveillance and effective treatment, and provide an approach to disease classification. We conclude that in order to progress our understanding of cancer aetiology, a balanced approach integrating molecular measures into well-designed epidemiological studies is required.

Heavy Metals, Resting Metabolism Rates and Breeding Parameters in Two Populations of Black-Headed GullLarus ridibundusfrom the Industrially Polluted Areas of Upper Silesia, Poland

Black-headed Gulls breeding in the central part of the Upper Silesian Industrial Region (Katowice-Szopienice) in Southern Poland were compared with colony from less polluted area (Świerklaniec) situated 23 km away. Heavy metals: Cd, Pb, Zn and Cu — were determined in various organs of nestlings, fledglings and mature birds as well as egg yolks. Resting metabolic rates, hatching success and eggshell thickness were used as biomarkers of environmental exposure to industrial pollutants. The clutch size (2.97 versus 3.61) and hatching success (81.5% versus 87%) were lower in the colony from the more polluted site. The relatively high metal contents in the yolks indicate that off spring are only partially protected from toxic compounds. Gulls were able to regulate body contents of essential metals Zn and Cu, but Pb and Cd accumulated rapidly in the liver, kidneys and lungs of growing birds. Cd also accumulated in the ovaries at the similar levels as in the kidneys. Cd and Pb may have affected earlier stages of development when parents were foraged in a close vicinity of the heavily polluted area but once the young gulls had reached maturity they then foraged in distant areas. During this period they were exposed to pollutants in the same way as the gulls from the less polluted site. There was no growth impairment identified in gulls from the more polluted area, and their resting metabolism (RMR) calculated per unit of body weight was lower, indicating that energetic costs for detoxification were not as high.

Both the environment and genes are important for concentrations of cadmium and lead in blood.

Concentrations of cadmium and lead in blood (BCd and BPb, respectively) are traditionally used as biomarkers of environmental exposure. We estimated the influence of genetic factors on these markers in a cohort of 61 monozygotic and 103 dizygotic twin pairs (mean age = 68 years, range = 49-86). BCd and BPb were determined by graphite furnace atomic absorption spectrophotometry. Variations in both BCd and BPb were influenced by not only environmental but also genetic factors. Interestingly, the genetic influence was considerably greater for nonsmoking women (h(2) = 65% for BCd and 58% for BPb) than for nonsmoking men (13 and 0%, respectively). The shared familial environmental (c(2)) influence for BPb was 37% for men but only 3% for women. The association between BCd and BPb could be attributed entirely to environmental factors of mutual importance for levels of the two metals. Thus, blood metal concentrations in women reflect not only exposure, as previously believed, but to a considerable extent hereditary factors possibly related to uptake and storage. Further steps should focus on identification of these genetic factors and evaluation of whether women are more susceptible to exposure to toxic metals than men.

The Relationship Between Reproductive Status and Metallothionein mRNA Expression in the Common Killifish, Fundulus heteroclitus

Metallothionein (MT), a ubiquitously distributed cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals and may serve as a defense mechanism as well as a biomarker of environmental exposure to toxic metals. In this study, basal expression of MT mRNA was characterized in male and female killifish Fundulus heteroclitus during non-spawning and spawning seasons. Hepatic, gill, and intestinal MT mRNA (copy number per μg total RNA) was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and electrochemiluminescence (ECL). There were no observed differences in MT mRNA expression between male and female killifish during either season. The median MT copy numbers for both non-spawning and spawning killifish decreased in the order of liver>intestine>gill. In addition, there were no significant differences in liver, gill and intestine tissue MT mRNA copy numbers within each season. However, a significant increase in gill and intestine MT mRNA induction was observed in spawning compared to non-spawning killifish (p<0.05). While these results indicate a reproductive seasonal difference in Fundulus basal MT expression, this difference was irrespective of sex. The results of this study demonstrate the variability of basal MT mRNA expression in three killifish tissues and confirm the need for further research prior to the use of MT mRNA as a biomarker of xenobiotic exposure in Fundulus heteroclitus.

HPRT mutations in vivo in human CD 34 + hematopoietic stem cells

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.

Gas chromatography–electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene

Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 μm film thickness, β=320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 162°C, for 14.2 min; ramp by 30°C/min to 190°C, for 3.5 min; ramp by 30°C/min to 250°C, for 4 min; helium flow rate: 1.7 ml/min; detector and injector temperature: 300°C. The sample (1 μl) was injected with a split injection technique (split ratio 5:1). MHA recovery was >95% in the 0.5–20 μmol/l range; the limit of detection was <0.25 μmol/l; day-to-day precision, at 2 μmol/l, was Cv<10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban air pollution (airborne xylene ranging from 0.1 to 3.7 μmol/m 3). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 μmol/l vs. 8.1 μmol/l, p<0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 μmol/l vs. 6.9 μmol/l, p<0.001), and evidenced a relationship between MHAs ( y, μmol/mmol creatinine) and airborne xylene ( x, μmol/m 3) ( y=0.085+0.34 x; r=0.82, p<0.001, n=56). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.

Evaluation of Biomarkers of Environmental Exposures: Urinary Haloacetic Acids Associated with Ingestion of Chlorinated Drinking Water

A study was conducted to determine if DCAA and TCAA urinary excretion rates are valid biomarkers of chronic ingestion exposure to these disinfection by-products of chlorination of drinking water. Entire first morning urine voids, time-of-visit urine samples, and tap water samples were collected from 47 female subjects. In addition, a 48-h recall questionnaire was administered to determine the amounts and types of liquids ingested by each subject as well as other exposures that could lead to DCAA and TCAA urinary excretion. The TCAA excretion rate for the first morning urine samples was significantly correlated with the estimated 48-h TCAA ingestion exposure for 25 subjects whose ingestion exposures primarily occurred at home, while the DCAA excretion rate was not correlated with the DCAA ingestion exposure. Thus, urinary TCAA appears to be a valid biomarker of chronic ingestion exposure to TCAA from chlorinated water, while urinary DCAA is not. It is proposed that the difference in the biological half-lives between these two compounds is the rationale for this finding. The biological half-life of TCAA is longer than successive exposure intervals; thus TCAA accumulates until it reaches a steady state. The half-life of DCAA is shorter than successive exposure intervals; thus DCAA is almost completely meta- bolized following an exposure and is eliminated from the body. This study suggests that biological half-life, exposure interval, and sample collection interval should be considered in selecting biomarkers and designing studies to validate them.

Environmental and urinary reference values as markers of exposure to hydrocarbons in urban areas.

A study using individual dosimetry to evaluate the daily inhaled dose of sixteen aromatic and aliphatic hydrocarbons in three groups of primary school children, living in three Italian towns with 50,000 inhabitants or less, (Treviglio-Lombardy; Poggibonsi-Tuscany; Valenza-Piedmont) is presented. The simultaneous use of two passive samplers (radial diffusion) for each child, for a 24 h period, determined both the indoor and indoor + outdoor environmental reference concentrations. We measured the urinary levels of benzene, toluene, ethylbenzene and xylenes for each child and hence determined the urinary reference values for BTEX. We also considered the possibility of using benzene in urine as a biomarker of environmental exposure of the general population to this xenobiotic. We evaluated how both the environmental and biological measures were influenced by the presence of smokers in the surveyed children's houses. For the group of children living in Poggibonsi, we considered the influence of the living area and the traffic density on environmental concentrations of benzene (indoor and indoor + outdoor).

Air toxics: biomarkers in environmental applications--overview and summary of recommendations.

On April 27-28, 1995, the National Urban Air Toxics Research Center hosted a Symposium on Air Toxics: Biomarkers in Environmental Applications. The purpose of the symposium was to define the current state of the art in the application of biomarkers for environmental exposures to benzene, toluene, styrene, 1,3-butadiene, polycyclic aromatic hydrocarbons, manganese, and chromium. Sensitive, specific, and cost-effective biomarkers of exposure, effect, and susceptibility may greatly improve our knowledge of the human health impacts of air toxics exposures. Presentations were made on the first day that provided state-of-the-art background for the need, use, and ethical considerations in biomarker research and applications. In workshops held during the symposium, a number of recommendations were made regarding the use and need for additional research with biomarkers. In general, the sensitivity and specificity of biomarkers for environmental exposures, need to be improved. Reliable effect and susceptibility biomarkers are not available for these compounds. More research needs to be done to establish and evaluate biomarkers, linked to health effects through mechanistic studies, at environmentally relevant exposure levels.

Quantification of t(14;18) in the lymphocytes of healthy adult humans as a possible biomarker for environmental exposures to carcinogens.

A t(14;18) chromosomal translocation is found in approximately 85% of follicular lymphomas by both cytogenetic and molecular analyses. This rearrangement deregulates expression of the bcl-2 proto-oncogene by translocation into the immuno-globulin heavy chain locus and is probably mediated by illegitimate V(D)J recombination. We have developed a quantitative nested PCR method for detecting this event in lymphocytes of healthy individuals. Genomic DNA is purified from peripheral blood lymphocytes, and 2.5 microg (representing 4 X 10(5) cells) are amplified with translocation-specific primers under conditions in which a single copy, if present, will give a detectable PCR product. Multiple replicates are analyzed for each individual, and Poisson statistics are then used to estimate the translocation mutant frequency. We have examined lymphocyte DNA from 34 healthy individuals by this assay and found the frequency of cells with t(14;18) to range from <0.8-96X10(-7). The molecular nature of the translocations has been investigated by determining the DNA sequence at the translocation junctions. In several individuals, multiple isolates of the same translocation event were recovered, indicating that the cell with the original translocation had undergone clonal expansion. In addition, multiple independent translocations were shown to occur within an individual. Since this translocation appears to be one step in the progression of a normal cell to a cancer cell, this assay may have utility as an effects biomarker for environmental carcinogen exposure.

Improved high-performance liquid chromatographic method for the determination of polycyclic aromatic hydrocarbon metabolites in human urine.

A reversed-phase HPLC method with fluorescence detection was evaluated for utility in determination of urinary metabolites of polycyclic aromatic hydrocarbons as biomarkers of environmental exposure. The method, which was developed for use in studies of high-level occupational exposure, was found to be unreliable for relatively low-level environmental exposures. The method was modified to include quantitation by standard addition in order to compensate for matrix effects at levels as low as 0.1 ng/ml. The standard addition modification increased both qualitative and quantitative performance, with recovery of 1-hydroxypyrene spikes improved from 164% to 114% at 0.36 ng/ml. The modified method was successfully applied in an environmental exposure study.

Induction of cytochrome P450 and other drug metabolizing enzymes in rat liver following dietary exposure to Aroclor 1254

Selected drug metabolizing activities were measured in female F344 NCr rats exposed to graded dietary concentrations of Aroclor 1254 (1 to 1000 ppm) for 7 days or to lower concentrations of Aroclor (1 to 10 ppm) for up to 28 days. Following the 7-day exposure, the hepatic O-dealkylation of ethoxyresorufin (ETR), mediated primarily by cytochrome P450IA, was increased 60-, 10-, and 4-fold by 33, 10, and 3 ppm Aroclor, respectively. In rats exposed to 10 and 3 ppm Aroclor for 28 days, this activity was increased approximately 30- and 10-fold, respectively. Hepatic ETR O-dealkylase activities correlated with Aroclor concentrations in the livers of exposed rats ( r = 0.99, p < 0.01). Although the O-dealkylation of benzyloxyresorufin was highly increased by 7-days dietary exposure to 1000 ppm Aroclor, the levels of Aroclor necessary for detection of induction were substantially higher than those required for detection of ETR O-dealkylase induction. Examination of the non-P450-mediated drug metabolizing activities, epoxide hydrolase and DT-diaphorase, similarly showed limited (∼ 10-fold) increases. In contrast, aldehyde dehydrogenase (benzaldehyde, NADP +) activity was highly increased (>40-fold) at 1000 ppm, however this activity was increased to only a limited extent at lower Aroclor concentrations (e.g., ∼ 3-fold at 33 ppm). These results support the potential use of cytochrome P450 activities as potential biomarkers for environmental exposure to PCBs and related compounds.