J.C.S. Kleinjans

Consumption of drinking water with high nitrate levels causes hypertrophy of the thyroid

We studied the effect of nitrate contamination of drinking water on volume and function of the thyroid in human populations exposed to different nitrate levels in their drinking water. Two sets of low and medium (tap) water, respectively medium and high (well) water nitrate exposure groups were compared. Drinking of nitrate-contaminated water was dose-dependently related with 24-h urinary nitrate excretion and salivary nitrate levels. No iodine deficiency was observed in any of the nitrate exposure groups. A dose-dependent difference in the volume of the thyroid was observed between low and medium vs. high nitrate exposure groups, showing development of hypertrophy at nitrate levels exceeding 50 mg/l. An inverse relationship was established between the volume of the thyroid and serum thyroid stimulating hormone (TSH) levels.

Nitrate contamination of drinking water: relationship with HPRT variant frequency in lymphocyte DNA and urinary excretion of N-nitrosamines.

We studied peripheral lymphocyte HPRT variant frequency and endogenous nitrosation in human populations exposed to various nitrate levels in their drinking water. Four test populations of women volunteers were compared. Low and medium tap water nitrate exposure groups (14 and 21 subjects) were using public water supplies with nitrate levels of 0.02 and 17.5 mg/l, respectively. Medium and high well water nitrate exposure groups (6 and 9 subjects) were using private water wells with mean nitrate levels of 25 and 135 mg/l, respectively. Higher nitrate intake by drinking water consumption resulted in a dose-dependent increase in 24-hr urinary nitrate excretion and in increased salivary nitrate and nitrite levels. The mean log variant frequency of peripheral lymphocytes was significantly higher in the medium well water exposure group than in the low and medium tap water exposure groups. An inverse correlation between peripheral lymphocyte labeling index and nitrate concentration of drinking water was observed. Analysis of N-nitrosamine in the urine of 22 subjects by gas chromatography-mass spectrometry revealed the presence of N-nitrosopyrrolidine in 18 subjects. Analysis of the mutagenicity of well water samples showed that a small number of the well water samples were mutagenic in the Ames Salmonella typhimurium test after concentration over XAD-2 resin. In conclusion, consumption of drinking water, especially well water, with high nitrate levels can imply a genotoxic risk for humans as indicated by increased HPRT variant frequencies and by endogenous formation of carcinogenic N-nitroso compounds from nitrate-derived nitrite. ImagesFigure 1.Figure 2.Figure 3.

Modulation of DNA and protein adducts in smokers by genetic polymorphisms in GSTM1,GSTT1, NAT1 and NAT2.

The formation of DNA and protein adducts by environmental pollutants is modulated by host polymorphisms in genes that encode metabolizing enzymes. In our study on 67 smokers, aromatic-DNA adduct levels were examined by nuclease P1 enriched 32P-postlabelling in mononuclear blood cells (MNC) and 4-aminobiphenyl-haemoglobin adducts (4-ABP-Hb) by gas chromatography-mass spectroscopy. Genetic polymorphisms in glutathione S-transferase M1 (GSTM1), T1 (GSTT1) and N-acetyl-transferase 1 (NAT1) and 2 (NAT2) were assessed by polymerase chain reaction-based methods. DNA adduct levels, adjusted for the amount of cigarettes smoked per day, were higher in GSTM1(-/-) individuals (1.30 +/- 0.57 adducts per 108 nucleotides) than in GSTM1(+) subjects (1.03 +/- 0.56, P = 0.05), higher in NAT1 slow acetylators (1.58 +/- 0.54) than in NAT1 fast acetylators (1.11 +/- 0.58, P = 0.05) and were also found to be associated with the NAT2 acetylator status (1.29 +/- 0.64 and 1.03 +/- 0.46, respectively, for slow and fast acetylators, P = 0.06). An effect of GSTT1 was only found in combination with the NAT2 genotype; individuals with the GSTT1(-/-) and NAT2-slow genotype contained higher adduct levels (1.80 +/- 0.68) compared to GSTT1(+)/NAT2 fast individuals (0.96 +/- 0.36). Highest DNA adduct levels were observed in slow acetylators for both NAT1 and NAT2 also lacking the GSTM1 gene (2.03 +/- 0.17), and lowest in GSTM1(+) subjects with the fast acetylator genotype for both NAT1 and NAT2 (0.91 +/- 0.45, P = 0.01). No overall effects of genotypes were observed on 4-ABP-Hb levels. However, in subjects smoking less than 25 cigarettes per day, 4-ABP-Hb levels were higher in NAT2 slow acetylators (0.23 +/- 0.10 ng/g Hb) compared to fast acetylators (0.15 +/- 0.07, P = 0.03). These results provide further evidence for the combined effects of genetic polymorphisms in GSTM1, GSTT1, NAT1 and NAT2 on DNA and protein adduct formation in smoking individuals and indicate that, due to the complex carcinogen exposure, simultaneous assessment of multiple genotypes may identify individuals at higher cancer risk.

Assessment of mutagenic activity of repeatedly used deep-frying fats

Mutagenic activity of repeatedly used deep-frying fats was evaluated in relation to chemical characteristics. Deep-frying fat samples were collected from local restaurants and snack bars after sensory indication of abuse. A total of 20 deep-frying fat samples and 2 unused control fat samples was tested. Fat samples were fractionated into non-polar and polar compounds by column chromatography. Amounts of polar compounds obtained ranged from 2% (by weight) for unused fat to 44% for used deep-frying fat. Levels of di- and polymeric triglycerides (DPTG) were determined using gel-permeation chromatography. DPTG concentrations of 13 used deep-frying fat samples exceeded the threshold level of 10% above which fats are rejected for use. In addition thiobarbituric acid-reactive substances (TBA-RS) were measured. Amounts of TBA-RS were just above detection levels for most fat samples. Five used fat samples, however, contained relatively high concentrations of TBA-RS, ranging from 82 to 177 nmoles malondialdehyde/g. Non-polar and polar fractions were screened for mutagenic activity using the Ames mutagenicity assay. Mutagenic activity was found predominantly in polar fractions at doses higher than 1 mg/plate in strains TA97, TA100 and TA104, variously with and without metabolic activation. The highest number of mutagenic samples was detected by strain TA97, which appeared to be most sensitive. Some samples exhibited toxic effects. Chromatography blanks, consisting of solvents processed according to the same procedures as used for fat samples, were not mutagenic. Mutagenic activity was also detected in polar material obtained from unused frying fat. Non-polar fractions of unused frying fats showed no mutagenicity. A frying experiment carried out under laboratory conditions indicated that during repeated and prolonged use of deep-frying fat mutagenic polar substances were formed. Fat samples taken after 20 and 40 h of frying contained increasing amounts of polar compounds. Mutagenic activity was highest after 20 h of frying but was slightly decreased after 40 h of frying. At this stage, however, mutagens also appeared in the non-polar fraction. Mutagenic activity of polar fractions of used deep-frying fats in strain TA97 was positively correlated with levels of TBA-RS, which may indicate the involvement of lipid oxidation products in mutagenicity of used deep-frying fats. No significant correlations were found with other chemical characteristics. In the process of deep-fat frying numerous degradation products are formed, which may include mutagenic heterocyclic amines and other pyrolysates.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitrate contamination of drinking water: evaluation of genotoxic risk in human populations.

Nitrate contamination of drinking water implies a genotoxic risk to man due to the endogenous formation of carcinogenic N-nitroso compounds from nitrate-derived nitrite. Thus far, epidemiological studies have presented conflicting results on the relation of drinking water nitrate levels with gastric cancer incidence. This uncertainty becomes of relevance in view of the steadily increasing nitrate levels in regular drinking water supplies. In an attempt to apply genetic biomarker analysis to improve the basis for risk assessment with respect to drinking water nitrate contamination, this study evaluates peripheral lymphocyte chromosomal damage in human populations exposed to low, medium, and high drinking water nitrate levels, the latter being present in private water wells. It is shown that nitrate contamination of drinking water causes dose-dependent increases in nitrate body load as monitored by 24-hr urinary nitrate excretion in female volunteers, but this appears not to be associated with peripheral lymphocyte sister chromatid exchange frequencies.

Estimate of the maximal daily dietary intake of butylated hydroxyanisole and butylated hydroxytoluene in The Netherlands.

The daily dietary intake of the phenolic antioxidants butylated hydroxyanisole (BHA) and/or butylated hydroxytoluene (BHT) was estimated using data obtained from a nationwide dietary record survey carried out in The Netherlands in 1987/1988. The estimates were based on the fat content of selected food categories and their respective maximum permitted levels of BHA and/or BHT. The results indicate that it is unlikely that the current acceptable daily intake for BHA (0-0.05 mg/kg body weight) is surpassed, even in individuals with an extremely high caloric intake, except in extreme cases in 1-6-year-olds. However, it cannot be excluded that the acceptable daily intake for BHT (FAO/WHO: 0-0.125 mg/kg; EEC: 0-0.05 mg/kg) is exceeded in all age and sex groups, but particularly in children aged 1-6 years.

Disposition of single oral doses of butylated hydroxyanisole in man and rat

The kinetics and metabolism of butylated hydroxyanisole (BHA) have been compared between man and rats. Oral doses of 2, 20 or 200 mg BHA/kg body weight were administered to male Wistar rats and a single oral dose of 0.5 mg/kg body weight was administered to human volunteers (non-smoking males). Following oral administration of 2 or 20 mg BHA/kg body weight to rats, no plasma BHA profiles were observed, whereas at the 200 mg BHA/kg body weight dose level plasma BHA peak concentrations between 100 and 400 ng/ml were detected. Plasma BHA peak levels and the area under the curve show that the application of 15% polyethylene glycol-400 as the vehicle produced significantly lower values compared with those obtained using the vehicles, salad dressing, corn oil and dimethylsulphoxide. In man, oral administration of 0.5 mg BHA/kg body weight dissolved in corn oil gave plasma BHA peak concentrations of greater value than 100 ng/ml (range 53 to 255 ng/ml). In rats, 24 hr after dosing 2, 20 or 200 mg BHA/kg body weight the mean BHA concentrations in adipose tissue ranged from 0.7 to 6.8 micrograms/g. In man and rats, BHA was O-demethylated to tert-butylhydroquinone (TBHQ). This is the first study to report that TBHQ is an in vivo metabolite of BHA in rats. Within 4 days following oral administration the total recovery of BHA in the urine and faeces of man (0.5 mg BHA/kg body weight) and rats (200 mg BHA/kg body weight) was 49 +/- 7% and 95 +/- 10% (mean +/- SD) respectively. In rats, BHA was excreted in the urine as free BHA (2%), conjugated BHA (48%) and conjugated TBHQ (9%) and in the faeces as free BHA (36%). In man, BHA was excreted in the urine mainly as conjugated BHA (39%) together with smaller amount of conjugated TBHQ (9%); no free BHA was found in the urine or faeces. In man and rats only the fraction of BHA excreted in urine as conjugates of BHA and TBHQ was qualitatively and quantitatively comparable. Results in this study indicate a considerable difference in the biological fate of BHA following oral administration of high and low doses of BHA in rat and man, respectively.

Genotoxicity of coal fly ash, assessed in vitro in Salmonella typhimurium and human lymphocytes, and in vivo in an occupationally exposed population

Fly ash as a product of coal combustion is known to contain various mutagenic substances, but genotoxic properties, especially of the particular (larger-size) fly ash fraction which is electrostatically precipitated (ESP) in the energy plant, have hardly been investigated. While smaller-size fly ash particles escape through the stack during powder coal combustion, the ESP fraction is collected and used for the manufacturing, for instance according to the Lytag process, of secondary products which can serve several construction purposes. Since fly ash as well as fly ash products are generally introduced into the human environment, a study of possible genotoxic effects to human DNA is indicated. Mutagenic properties of ESP fly ash, as well as of the Lytag product, were investigated by means of the Salmonella microsome assay. The capacity to cause human chromosome damage of both ESP fly ash and Lytag dust was studied in vitro by application of the sister-chromatid exchange (SCE) test using human lymphocytes. Furthermore, effects of ESP fly ash/Lytag dust on the incidence of SCE in peripheral lymphocytes in vivo were measured in an occupationally exposed, male population, using individually matched employees from a flour-processing industry as the control population. It is demonstrated that ultrasonically treated DMSO extracts of ESP fly ash are slightly mutagenic to Salmonella tester strains TA97 and TA102. Lytag dust is effective in inducing reversions in all tester strains. Furthermore, it appeared that both compounds significantly increase the SCE frequency of human lymphocytes after incubation in vitro in comparison to non-exposed cells. Also, peripheral lymphocytes of the occupationally exposed population show a considerably higher incidence of SCE than the control population. Major disturbing factors in assessing the effects of occupational exposure to fly ash/Lytag dust on lymphocyte SCE frequency appeared to be smoking behavior and alcohol consumption. It is concluded that exposure to fly ash from powder coal combustion implies a moderate genotoxic risk to man.

Dietary acrylamide intake and risk of breast cancer in the UK women's cohort

Background:No studies to date have demonstrated a clear association with breast cancer risk and dietary exposure to acrylamide.Methods:A 217-item food frequency questionnaire was used to estimate dietary acrylamide intake in 33 731 women aged 35–69 years from the UK Womens Cohort Study followed up for a median of 11 years.Results:In all, 1084 incident breast cancers occurred during follow-up. There was no evidence of an overall association between acrylamide intake and breast cancer (hazard ratio=1.08 per 10 μg day−1, 95% CI: 0.98–1.18, Ptrend=0.1). There was a suggestion of a possible weak positive association between dietary acrylamide intake and premenopausal breast cancer after adjustment for potential confounders (hazard ratio=1.2, 95% CI: 1.0–1.3, Ptrend=0.008). There was no suggestion of any association for postmenopausal breast cancer (hazard ratio=1.0, 95% CI: 0.9–1.1, Ptrend=0.99).Conclusions:There is no evidence of an association between dietary acrylamide intake and breast cancer. A weak association may exist with premenopausal breast cancer, but requires further investigation.

Relationship between Radical Generation by Urban Ambient Particulate Matter and Pulmonary Function of School Children

The mechanisms by which particulate matter (PM) produces adverse effects on the respiratory system, such as pulmonary dysfunction in children, are largely unknown. However, oxidative stress is thought to play an important role. Various chemical compounds in ambient particulate matter, including transition metals and aromatic organic compounds, may contribute to adverse effects through intrinsic generation of reactive oxygen species (ROS). It was hypothesized that ROS generation by PM, as determined through electron spin resonance (ESR) spectroscopy, may be negatively associated with pulmonary function in school children. PM2.5, PM10, and total suspended particulates (TSP) were sampled at the playgrounds of six elementary schools in the city of Maastricht, the Netherlands. All children (8–13 yr) from the six schools were asked to undergo spirometry. Multivariate linear regression models were constructed to evaluate associations between oxygen radical formation by PM and lung function. The radical-generating capacity per microgram PM correlated negatively to forced expiratory volume in 1 s (FEV1) and forced expiratory flow at 50% (FEF50%) of forced vital capacity (FVC). The data indicate that chemical features that contribute to intrinsic generation of ROS may be relevant for PM risk assessment.