Maternal drinking water arsenic exposure and perinatal outcomes in inner Mongolia, China.

BACKGROUND: Bayingnormen is a region located in western Inner Mongolia China, with a population that is exposed to a wide range of drinking water arsenic concentrations. The relationship between maternal drinking water arsenic exposure and perinatal endpoints (term birth weight, preterm birth, stillbirth and neonatal death) in this region was evaluated in this study. METHODS: An analysis was conducted of all singleton deliveries in a defined geographical area of Inner Mongolia from December 1996 to December 1999 (n=9890). Outcome and covariate data were abstracted from prenatal care records. Exposure was based on well-water measures for the maternal subvillage. Mean birth weight at term was compared across four arsenic categories using analysis of covariance. ORs for stillbirth, preterm birth and neonatal death were estimated by logistic regression with arsenic exposure dichotomised at 50 microg/l. RESULTS: Term birth weight was 0.05 kg higher (95% CI 0.02 to 0.08) in the highest exposure category (>100 microg/l) compared to the reference (below limit of detection to 20 microg/l). Arsenic >50 microg/l was associated with an increased risk of neonatal death (OR 2.01, 95% CI 1.12 to 3.59). No relationship was found between maternal arsenic exposure and preterm or stillbirth delivery. CONCLUSIONS: At the levels observed in our study, arsenic does not appear to contribute to adverse birth outcomes. Exposure may play a role in neonatal death; however, the neonatal death rate in this population was low and this potential association merits further research.

Pregnancy and perinatal health in Inner Mongolia, China, 1996-1999.

OBJECTIVE: To obtain descriptive measures of maternal and perinatal health in the Ba Men Region of Inner Mongolia, China. METHODS: Data collected from the Examination Chart for Pregnant Women for approximately 22,000 pregnancies in a three-county area of Inner Mongolia, China from December 1, 1996 through December 31, 1999 were analyzed for maternal, perinatal, and neonatal outcomes. RESULTS: Compared to selected developing countries, a higher percentage of women (99%) in this region received at least one prenatal care visit. This region was also characterized by a low percentage of low birthweight (<2.5 kg) infants (1%) and neonatal mortality rate (5 deaths per 1000 live births). CONCLUSIONS: Maternal and neonatal health outcomes in this region of Inner Mongolia were better than those in selected developing countries.

Human nails as a biomarker of arsenic exposure from well water in Inner Mongolia: comparing atomic fluorescence spectrometry and neutron activation analysis.

Arsenic (As) is found naturally in the geological strata within the Ba Men Region of Inner Mongolia, China. A study was conducted to compare the total As measurements from two analytical techniques: instrumental neutron activation analysis (INAA) and atomic fluorescence spectrometry (AFS), and to verify nails as an exposure biomarker in this population. In 1999, nail and water samples were collected in a pilot study. Fingernails and toenails were pooled from 32 participants and analysed for total As by both INAA and AFS. Mean nail As values were 14.8+/-2.4 and 19.4+/-2.8 microg g-1 (+/-SEM) for INAA and AFS, respectively. Results from these two methods were significantly correlated (r=0.93, p<0.0001). In 2000, a second study was conducted and INAA was used to measure total As in toenails from 314 Ba Men residents. Well water samples were collected from 121 households and analysed by AFS. A significant correlation was observed between toenail and well water As (r=0.84, p<0.0001). Based on the results, INAA was significantly correlated with AFS and proved to be a reliable measure of nail As levels. In this population, toenail samples are a useful internal As exposure biomarker from drinking water sources.

Analyses of micronuclei in exfoliated epithelial cells from individuals chronically exposed to arsenic via drinking water in inner Mongolia, China.

The groundwater in Bayingnormen (Ba Men), located in Central West Inner Mongolia, China, is naturally contaminated with arsenic at concentrations ranging from 50 microg/L to 1.8 mg/L. Various adverse health effects in this region, including cancer, have been linked to arsenic exposure via drinking water. A pilot study was undertaken to evaluate frequencies of micronuclei (MN), as measures of chromosomal alterations, in multiple exfoliated epithelial cell types from residents of Ba Men chronically exposed to arsenic via drinking water. Buccal mucosal cells, airway epithelial cells in sputum, and bladder urothelial cells were collected from 19 residents exposed to high levels of arsenic in drinking water (527.5 +/- 24 microg/l), and from 13 control residents exposed to relatively low levels of arsenic in drinking water (4.4 +/- microg/L). Analytical results from these individuals revealed that MN frequencies in the high-exposure group were significantly elevated to 3.4-fold over control levels for buccal and sputum cells, and to 2.7-fold over control for bladder cells (increases in MN frequency significant at p < .001 for buccal cells; p < .01 for sputum cells; p < .05 for bladder cells). When smokers were excluded from high-exposure and control groups the effects of arsenic were observed to be greater, although only in buccal and sputum cells; approximately 6-fold increases in MN frequency occurred in these tissues. The results indicate that residents of Ba Men chronically exposed to high levels of arsenic in drinking water reveal evidence of genotoxicity in multiple epithelial cell types; higher levels of induced MN were observed in buccal and sputum cells than in bladder cells.

Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustion emissions.

We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G --> T transversions at either KRAS (86%) or TP53 (76%), (b) clustered at the G-rich codons 153-158 of TP53 (33%), and (c) had 100% of the guanines of the G --> T transversions on the nontranscribed strand. This mutation spectrum is consistent with an exposure to polycyclic aromatic hydrocarbons, which are the primary component of the smoky coal emissions. These results show that mutations in the TP53 and KRAS genes can reflect a specific environmental exposure.

p53 gene expression in relation to indoor exposure to unvented coal smoke in Xuan Wei, China.

Lung cancer mortality rates in Xuan Wei County, which are among the highest in China, have previously been associated with exposure to indoor emissions from burning smoky coal. To determine if this association is stronger among lung cancer patients with abnormal expression of p53, we performed a population-based case-control study. Ninety-seven newly diagnosed lung cancer patients and 97 controls, individually matched by age, sex, and home fuel type, were enrolled. We used immunocytochemical methods to assess p53 protein accumulation in exfoliated tumor cells isolated from sputum samples. As expected, the amount of lifetime smoky coal use was associated with an overall increase in lung cancer risk. Compared with subjects who used less than 130 tons of smoky coal during their lifetime, the odds ratios (OR) for lung cancer were 1.48 (95% confidence interval [CI], 0.73 to 3.02) for subjects exposed to 130 to 240 tons, and 3.21 (95% CI, 1.23 to 9.03) for subjects who used more than 240 tons of smoky coal (P for trend 0.01). The effect was due almost exclusively to the pattern in women, almost all of whom were nonsmokers. Further, among highly exposed women, the association was substantially larger and achieved statistical significance only among patients with sputum samples that were positive for p53 overexpression (OR, 18.72; 95% CI, 1.77 to 383.38 vs OR, 4.80; 95% CI, 0.66 to 43.87 for p53-negative cases). This study suggests that exposure to the combustion products of smoky coal in Xuan Wei is more strongly associated with women who have lung cancer accompanied by p53 protein overexpression in exfoliated tumor cells.

DNA damage in buccal epithelial cells from individuals chronically exposed to arsenic via drinking water in Inner Mongolia, China.

The purpose of this pilot study was to assess DNA damage in buccal cells from individuals chronically exposed to arsenic via drinking water in Ba Men, Inner Mongolia. Buccal cells were collected from 19 Ba Men residents exposed to arsenic at 527.5 +/- 23.7 micrograms/L (mean +/- SEM) and 13 controls exposed to arsenic at 4.4 +/- 1.0 micrograms/L. DNA fragmentation by the DNA ladder and TUNEL assay were used to detect DNA damage in buccal cells. In the DNA ladder assay, 89% (17/19) of the arsenic-exposed group showed < 100 bp DNA fragments, in contrast to 15% (2/13) of the controls (p < 0.0001). For the TUNEL assay, the mean frequencies of positive cells were higher in the exposed group (15.1%) than in the controls (2.0%) (p < 0.0001). This study showed that high arsenic exposure via drinking water resulted in DNA damage and DNA fragmentation in buccal cells thus may be an appropriate biomarker for assessing chronic effects of arsenic in humans. A study investigating DNA fragmentation from the individuals with low levels of arsenic exposure in this population is in progress.

Detection and quantification of depurinated benzo[a]pyrene-adducted DNA bases in the urine of cigarette smokers and women exposed to household coal smoke.

Polycyclic aromatic hydrocarbons (PAH) are metabolized to electrophiles that can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAH-treated mouse skin. Depurinating adducts account for 71% of all DNA adducts formed in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls for the presence and quantities of the depurinated BP-adducted DNA bases, 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade). Since these adducted bases originate from reaction of the BP radical cation with double-stranded DNA and not with RNA or denatured DNA, their presence in urine is indicative of DNA damage. Urine samples were fractionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis with laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to coal smoke, but were not detected in urine from the 13 control subjects. Concentrations were estimated to be 60-340 and 0.1-0.6 fmol/mg of creatinine equivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20-300 times the concentration of BP-6-N7Ade in the urine of coal smoke-exposed women, but was not detected in the urine of cigarette smokers. This difference may be due to the remarkably different BP exposures experienced by the two groups of PAH-exposed individuals. These results justify more extensive studies of depurinated BP-adducted DNA bases as potential biomarkers of PAH-associated cancer risk.

Indoor coal combustion emissions, GSTM1 and GSTT1 genotypes, and lung cancer risk: a case-control study in Xuan Wei, China.

The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been associated with exposure to indoor smoky coal emissions that contain high levels of polycyclic aromatic hydrocarbons. This risk may be modified by variation in metabolism genes, including GSTM1, which encodes an enzyme known to detoxify polycyclic aromatic hydrocarbons. To investigate the relationship between GST genotypes and lung cancer risk in Xuan Wei County, we analyzed GSTM1 and GSTT1 genotypes in a population-based case-control study. A total of 122 lung cancer patients and 122 controls, individually matched by age, sex, and home fuel type, were studied. Compared to subjects who used less than 130 tons of smoky coal during their lifetime, heavier users (> or =130 tons) had a 2.4-fold (95% confidence interval, 1.3-4.4) increased risk of lung cancer. The GSTM1-null genotype was associated with a 2.3-fold (95% confidence interval, 1.3-4.2) increased risk of lung cancer. Furthermore, there was some evidence that smoky coal use was more strongly associated with lung cancer risk among GSTM1-null versus GSTM1-positive individuals. In contrast, the GSTT1 genotype was not significantly associated with lung cancer risk. Our data suggest that the GSTM1-null genotype may enhance susceptibility to air pollution from indoor coal combustion emissions.

Detection of p53 protein accumulation in sputum and lung adenocarcinoma associated with indoor exposure to unvented coal smoke in China.

Lung cancer in Xuan Wei (XW), China has been linked to exposure to unvented coal smoke and adenocarcinoma, especially bronchioloalveolar carcinoma, is most common. p53 mutations occur commonly in lung cancers and usually generate detectable levels of p53 protein accumulation. Sputum is noninvasive to collect and ideal for screening p53 abnormalities. p53 protein accumulation was detected by immunohistochemistry in lung tumors and sputa from XW lung cancer patients to determine (1) the role of p53 in lung pathogenesis, and (2) feasibility of detecting p53 protein accumulation in sputum, p53 protein accumulation was detected in 73% (22/30) of lung adenocarcinomas from XW females exposed to coal emissions and significantly higher than the control cases (33%, p < 0.05). In sputum, we detected p53 overexpression in tumor cells in 54% (13/24) of XW cases and also in dysplastic cells (50% or 4/8). These findings suggest that p53 abnormalities is important in XW lung cancer etiology.

A sensitive immunofluorescence assay for detection of p53 protein accumulation in sputum.

p53 mutations are common genetic alterations in lung cancers and usually result in p53 protein accumulation in tumor cells. Sputum is noninvasive to collect and ideal for screening p53 abnormalities. This study was to determine the feasibility of detecting p53 protein accumulation in sputum cells using an immunofluorescence assay. Sputum samples were collected from 58 Chinese subjects, including 16 lung cancer cases from Xuan Wei (XW) County exposed to coal smoke, 25 mostly tobacco smoke-related cases from Zhengzhou City, and 17 XW controls. The p53 protein accumulation in tumor cells and some atypical cells was detected in 56% (9/16) of the coal smoke-exposed XW cases, 44% (11/25) of the Zhengzhou cases and none (0/17) in controls. The p53+ tumor cells were confirmed morphologically by Papanicolaou staining after the p53 immunofluorescence assay. This is the first report of successfully using an immunofluorescence assay to detect p53 protein accumulation in sputum.

Multifocal accumulation of p53 protein in esophageal carcinoma: evidence for field cancerization.

A systematic characterization of the cancerization field of esophageal carcinoma based on p53 protein accumulation has not been reported previously. The present report presents such a study based on 50 specimens of esophageal squamous-cell carcinoma from northern China. To gain insight into the etiology of this disease among the 50 subjects, DNA was analyzed for a polymorphism of the aldehyde dehydrogenase-2 (ALDH2) gene, which has been associated with increased risk for esophageal cancer among alcohol-consuming patients in Japan. However, the frequency of this polymorphism among our subjects, 30% (15/50), was within published control frequencies for this allele, suggesting that this allele may not play a role in the etiology of esophageal cancer in this northern Chinese population. Immuno-histochemical staining showed that 66% of the tumors were p53+. Of 420 pieces near or adjacent to p53+ tumors, p53+ cells were present among 64% of basal-cell hyperplasia (BCH), 70% of dysplasia (DYS) and 88% of carcinoma in situ (CIS). Of 216 pieces near or adjacent to p53- tumors, p53+ frequencies were 25% of BCH, 25% of DYS and 0% of CIS. The proportion of BCH cells that were p53+ decreased at increasing distance from the tumor (p = 0.006). The sporadic distribution of p53+ cells and the distribution and frequency of p53+ precursor lesions support the view that accumulation of p53 protein is multifocal and occurs in precursor lesions in early stages of esophageal carcinogenesis.

A sensitive color ELISA for detecting polycyclic aromatic hydrocarbon-DNA adducts in human tissues.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been determined by measurement of DNA adducts in human tissues. Competitive enzyme-linked immunosorbent assays (ELISAs) using antisera recognizing benzo[a]pyrenediol-epoxide-modified DNA (BPDE-I-DNA) and color of fluorescence endpoint detection have been used extensively for quantifying PAH-DNA adducts. The fluorescence ELISA (limit of detection 1 adduct/10(8) nucleotides) was previously reported to be more sensitive than the color ELISA (1/10(7)) for measuring PAH adducts (Santella et al. (1988) Carcinogenesis, 9, 1265-1269). However, the fluorescence assay has the disadvantages of greater variation among the replicates and higher background levels than the color assay. Using a newly developed antiserum against BPDE-I-DNA, we have modified the color of ELISA so that it has the same sensitivity as the fluorescence ELISA and requires only 33% of the sample quantity needed for the fluorescence ELISA. The modifications included preincubation of the antiserum with the samples, using microtiter plates with half-size, flat bottom wells, and optimizing the assay conditions. The improved color ELISA was used to analyze DNA samples from human autopsy tissues, including heart, lung, liver, kidney, spleen, pancreas and stomach from smokers and nonsmokers. With the exception of spleen and stomach, all tissues from smokers showed higher PAH-DNA adducts (ranging from 0.3 to 19.0 adducts/10(7) nucleotides) than the tissues from the nonsmokers (0.3 to 3.7 adducts/10(7) nucleotides) in two separate experiments. Among the tissues from smokers, heart showed the highest level of DNA adducts. This study demonstrates that a stable color ELISA with high sensitivity can be useful in assessing human exposure to PAH.

Human exposure and dosimetry of polycyclic aromatic hydrocarbons in urine from Xuan Wei, China with high lung cancer mortality associated with exposure to unvented coal smoke.

The lung cancer mortality rate in Xuan Wei (XW) county, China, is 5-fold the national average of China; the rate for women is the highest in China. Xuan Wei residents have been exposed to unvented coal or wood smoke during cooking or heating in homes. This study investigated indoor air exposure and dosimetry of polycyclic aromatic hydrocarbons (PAHs) in XW residents using smoky coal. Indoor air particles collected during cooking in four XW homes using smoky coal were analyzed for PAHs by GC/MS. Urine samples from 16 XW non-smoking women and six XW smoking men, eight Kunming non-smoking controls and four non-smoking Chinese American controls were analyzed for PAHs and hydroxy-PAHs by GC/MS. The results showed that XW residents were exposed to PAHs at occupational levels. The potent carcinogen, dibenzo[a,l] pyrene (4.9 +/- 1.3 micrograms/m3) was found in the indoor air of the XW homes. The levels of urinary hydroxy-PAH were higher than those of the parent compounds in most subjects, indicating that most PAHs were metabolized. In urine, the mean levels of 9-hydroxy BaP (BaP) and BaP are 1.5 +/- 0.5 mumol/mol creatinine and 0.5 +/- 0.3 microns/mol for XW men, 1.9 +/- 0.9 microns/mol and 0.5 +/- 0.3 microns/mol for XW women. In general, the levels of PAH metabolites in urine were higher in the XW residents than in Kunming and Chinese American controls; however only the concentrations of 9-hydroxy BaP in XW women showed statistically significant difference from the Kunming controls (P < 0.05 by ranking test). The mean levels of 3 methylated-PAHs analyzed were 4.8-fold higher than that of the parent PAHs in XW subjects. This is consistent with previous findings that alkylated PAHs are the major mutagens in the XW indoor air and may be etiologically important in XW lung cancer.

DNA adducts as biomarkers for assessing exposure to polycyclic aromatic hydrocarbons in tissues from Xuan Wei women with high exposure to coal combustion emissions and high lung cancer mortality.

The high lung cancer rate in Xuan Wei, China, is associated with smoky coal use in unvented homes, but not with wood or smokeless coal use. Smoky coal combustion emits higher polycyclic aromatic hydrocarbon (PAH) concentrations than wood combustion. This study used DNA adducts as biomarkers for human exposure to PAH from combustion emissions. DNA adducts were determined by enzyme-linked immunosorbent assays (ELISA) in placentas and peripheral and cord white blood cells (WBC) from Xuan Wei women burning smoky coal or wood and from Beijing women using natural gas. Color ELISA gave positive results in 58, 47, and 5% of the placentas from Xuan Wei women burning smoky coal without and with chimneys, and from Beijing women, respectively. Fluorescence ELISA indicated that 46, 65, 56, and 25% of placentas were positive from Xuan Wei women who lived in houses without and with chimneys, Xuan Wei women burning wood, and Beijing controls, respectively. Peripheral WBC samples were positive in 7/9, 8/9, and 3/9 for the Xuan Wei women who lived in houses without and with chimneys and Beijing women, respectively. PAH-DNA adducts were detected in a higher percentage of placentas from Xuan Wei women living in houses exposed to smoky coal or wood emissions than from those of the Beijing controls. No dose-response relationship was observed between the air benzo[alpha]pyrene concentrations and DNA adduct levels or percentage of detectable samples. The results suggest that DNA adducts can be used as a qualitative biomarker to assess human exposure to combustion emissions.

Mutagenicity of organic emissions from unvented kerosene heaters in a chamber study.

A study was conducted to assess the mutagenicity of semivolatile organics and particle-bound organics emitted from unvented kerosene space heaters. The units tested included a well-tuned radiant heater and a maltuned convective heater. The tests were conducted in a 27-m3 chamber with a prescribed on/off heater usage pattern. The organic emissions were collected on Teflon-coated glass filters backed by XAD-2 resin. The dichloromethane-extractable organics from both the filters and the XAD were analyzed for nitropolycyclic hydrocarbons using gas chromatography/mass spectrometry, and were bioassayed for mutagenicity in microsuspension assays using Salmonella typhimurium strains TA98 with and without S9 and TA98NR (a nitroreductase-deficient strain) without S9. The results showed that both the semivolatile and particle-bound organics emitted from the kerosene heaters were mutagenic, and the presence of nitropolycyclic hydrocarbons in these organic emissions substantiated these findings.

Mouse skin tumorigenicity studies of indoor coal and wood combustion emissions from homes of residents in Xuan Wei, China with high lung cancer mortality.

The rural Xuan Wei County, Yunnan Province, China, has an unusually high lung cancer mortality rate that cannot be attributed to tobacco smoke or occupational exposure. The lung cancer rate is associated with 'smoky' coal, in contrast to wood or 'smokeless' coal burned in unventilated homes. This study was conducted to characterize and compare mouse skin tumorigenicity of the coal and the wood combustion emissions and to link the animal data to human lung cancer. Indoor air particles (less than 10 microns) were collected from a central commune where the lung cancer mortality rate is high and smoky coal is the major fuel used and also from a south-western commune where lung cancer mortality rate is low and where wood or smokeless coal are the major fuels used. The organic extracts of these indoor air particles from smoky coal, smokeless coal and wood combustion were analysed for polycyclic aromatic hydrocarbons and assayed for skin tumor initiation activity and complete carcinogenicity in SENCAR mice. The results showed that the organic extract of the emission particles from smoky coal combustion is the most active in tumor initiation among the three combustion emission samples followed by smokeless coal and then wood. The organic extract of the particles from smoky coal combustion was shown to be a potent complete carcinogen, whereas the wood extract was relatively inactive as a complete carcinogen. The extract of particles from the smokeless coal combustion was not tested for complete carcinogenicity because of inadequate supply. Eighty-eight percent of the mice treated with the smoky coal extract showed carcinomas, averaging 1.1 carcinomas per tumor-bearing mouse at the end of the 77 week study. These findings were in agreement with the epidemiological data, which showed that the Xuan Wei residents using smoky coal as a major fuel in homes had a high lung cancer mortality rate. This study demonstrates that the results of the tumorigenicity assays in mice were in agreement with human lung cancer data.

The epidemiology of lung cancer in Xuan Wei, China: current progress, issues, and research strategies.

In Xuan Wei, a rural Chinese county of about one million people, females' annual lung cancer mortality is China's highest, and males' is among China's highest. Xuan Wei's very high indoor air pollution levels (sometimes exceeding 20 mg/m3), residentially stable population, relatively uncomplicated lifestyle, and wide geographic variation in lung cancer mortality render it highly amenable to quantitative, interdisciplinary investigation of chemical carcinogens due to indoor air pollution. To date, epidemiologic findings reveal a closer association of lung cancer with the indoor burning of "smoky" coal (as opposed to "smokeless" coal or wood) than with tobacco use or occupation. Current aerometric, chemical, and toxicologic findings tend to confirm this association, though the specific carcinogenic constituents of Xuan Wei indoor air pollution have not yet been determined. Chinese and American investigators are conducting interdisciplinary field and laboratory investigations to quantify the lung cancer risk attendant on indoor air pollution relative to other factors, to measure and compare the characteristics of pollution from different Xuan Wei fuels, to determine the relative etiologic importance of pollution composition and concentration, and to develop quantitative relationships between air pollution dose and lung cancer risk.

Lung cancer and indoor air pollution in Xuan Wei, China.

In Xuan Wei County, Yunnan Province, lung cancer mortality is among China's highest and, especially in females, is more closely associated with indoor burning of "smoky" coal, as opposed to wood or "smokeless" coal, than with tobacco smoking. Indoor air samples were collected during the burning of all three fuels. In contrast to wood and smokeless coal emissions, smoky coal emission has high concentrations of submicron particles containing mutagenic organics, especially in aromatic and polar fractions. These studies suggested an etiologic link between domestic smoky coal burning and lung cancer in Xuan Wei.