Urinary polycyclic aromatic hydrocarbons and monohydroxy metabolites as biomarkers of exposure in coke oven workers.

OBJECTIVES: To assess exposure to polycyclic aromatic hydrocarbons (PAHs) using 13 unmetabolised PAHs (U-PAHs) and 12 monohydroxy metabolites (OHPAHs) in urine, and to compare the utility of these biomarkers. METHODS: 55 male Polish coke oven workers collected urine spot samples after a workshift. U-PAHs (naphthalene, acenaphtylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene) were determined by automatic solid phase micro-extraction followed by gas chromatography/mass spectrometry (GC/MS). OHPAHs (1- and 2-hydroxynaphthalene, 2- and 9-hydroxyfluorene, 4-, 9-, 3-, 1- and 2-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene, 3-hydroxybenzo[a]pyrene) were determined, after liquid/liquid extraction and derivatisation, by GC/MS. RESULTS: U-PAHs from naphthalene to chrysene were found in 100% of samples, and heavier U-PAHs in 7-22% of samples. OHPAHs up to 1-hydroxypyrene were found in 100% of samples, while 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene were always below the quantification limit. Median naphthalene, phenanthrene, pyrene, chrysene and benzo[a]anthracene levels were 0.806, 0.721, 0.020, 0.032 and 0.035 microg/l, while hydroxynaphthalenes, hydroxyphenanthrenes and 1-hydroxypyrene levels were 81.1, 18.9 and 15.4 microg/l. For each chemical, the ratio between U-PAH and the corresponding OHPAH ranged from 1:26 to 1:1000. Significant correlations between logged values of U-PAHs and OHPAHs, between U-PAHs, and between OHPAHs were found, with Pearson's r ranging from 0.27 to 0.97. CONCLUSION: Current analytical techniques allow specific and simultaneous measurement of several urinary determinants of PAHs in humans. The results of these measurements support the use of U-PAHs as biomarkers of exposure and suggest the spectrum of chemicals to be investigated, including carcinogenic chrysene and benzo[a]anthracene, should be widened.

How to prevent immunological reactions in leprosy patients and interrupt transmission of Mycobacterium leprae to healthy subjects: two hypotheses.

The basis of World Health Organization strategy for leprosy elimination is that the only source and reservoir for infection are patients with the disease. It was assumed that multi drug therapy (MDT) would reduce transmission of Mycobacterium leprae, but there is no convincing evidence for this. Furthermore, even if MDT has been proved to be extremely effective against the infectious disease, a noticeable proportion of leprosy patients can suffer from immunologic hypersensitivity reactions which are now the most significant issue in the managements of the disease. In endemic areas it was found that: M. leprae survives outside human body; healthy individuals harbor M. leprae bacilli in nasal cavity and shed micro-organisms in environment; there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response. This disparate clinical, epidemiologic, and microbiologic evidence leads to the first hypothesis: that antigenic load in local tissues, sufficient to trigger the immune response, comes from external supply of M. leprae organisms. The hypothiocyanite anion (OSCN-) is generated in vivo by the reaction of thiocyanate with hydrogen peroxide catalyzed by lactoperoxidase. OSCN- is an antimicrobial oxidizing agent that prevents growth of bacteria, fungi, and viruses. OSNC- exists in lower airway secretions and lung has never been reported to be affected by leprosy. There is a sufficient concentration of OSCN- in the saliva, and accordingly mouth is rarely affected by leprosy. By contrast, the concentration of this compound is low or nil in nasal and lacrimal secretions and leprosy very often affects nose and eyes. The second hypothesis is that OSCN- may also protect from leprosy. Recently a method of OSCN- production, not involving enzymatic steps or use of toxic heavy-metal salts, has been patented. Studies on the susceptibility of M. leprae to hypothiocyanite could be carried out and, in case of positive results, the substance might be used in order to sterilize the nasal cavity of healthy carriers and prevent transmission of M. leprae to healthy subjects and to leprosy patients in whom it may trigger an immune response.

Assessment of urinary mutagens presence in a population of non smokers.

The paper presents the early results of a study involving a group of 312 non smoking and not professionally exposed subjects (144 males and 168 females) in order to evaluate the probable presence of urinary mutagens possibly derived from aspecific exposures. Urine samples were assayed by the Ames test on the YG1024 Salmonella typhimurium strain in the presence of S9 mix with plate incorporation method with preincubation. At the moment of sample collection, the subjects were invited to fill a questionnaire on their main characteristics and lifestyle. On the basis of laboratory data analysis, it emerged that, on 288 samples with a valuable mutagenic activity, 20 urinary extracts (8 of which were males and 12 were females) showed mutagenicity levels twice as much as spontaneous revertants. Diet and indoor exposure to passive smoking, fireplace and cooking fume exposure seemed to play a major role among the lifestyle behaviours investigated in generating positive mutagenic response with a statistically significant difference between positive and negative samples induction (Chi square, P = 0.0057 and P = 0.0168 respectively). After correction of induced revertants by means of creatinine excretion determination, it appeared that females, who had the higher mean urinary mutagenic activity, showed a mutagenicity level twice as much as men (364 +/- 491 revertants/mmole creatinine for males against 605 +/- 868 revertants/mmole creatinine in females, Mann-Whitney U-test, z = -3.97, P < 0.0001) possibly in consequence of their greater cooking fumes exposure. The study, that carefully evaluated the characteristics of involved subjects, reveals the presence, even though modest, of mutagens in urine of an apparently not significantly exposed population. In addition, standardization of method leads to suppose little feasible a confounding influence of considered features. Moreover, it would be therefore rather interesting to study the effect of low exposure time persistence.

[Anti-B[a]PDE-DNA formation in lymphomonocytes of humans environmentally exposed to polycyclic aromatic hydrocarbons]. / Formazione dell'addotto anti-B[a]PDE-DNA nei linfomonociti nelle basse esposizioni ambientali ad idrocarburi policiclici aromatici.

[Anti-B[a]PDE-DNA formation in lymphomonocytes of humans environmentally exposed to polycyclic aromatic hydrocarbons] We are currently evaluating anti-benzo[a]pyrenediolepoxide-(B[a]PDE)-DNA adduct levels in lymphomonocytes of humans exposed to polycyclic aromatic hydrocarbons (PAHs) to validate this indicator of biologically effective dose in a surrogate tissue. The study protocol (October 2002-June 2005) implies: (a) a signed informed consent by each participant; (b) recruitment of 600 Padua municipal workers during visits at our outpatient clinic; (c) administration of a questionnaire regarding non occupational sources of PAH (B[a]P) exposure; (d) collection of blood (15 ml) and urine (200 ml) samples. Anti-B[a]PDE-DNA adduct levels in lymphomonocytes are detected by HPLC-fluorescence analysis. To date, 438 subjects have been examined (age range 20-62 years; 52% males). We found that: (i) anti-B[a]PDE-DNA adduct levels are significantly lower than those we previously found in coke-oven workers (N=95) occupationally exposed to high levels of PAHs (1.51 +/- 2.68 versus 4.07 +/- 3.78 anti-B[a]PDE-adduct/10(8) nucleotides, p < 0.001; 37% versus 97% positive subjects with > or =1 adduct/10(8) nucleotides; p < 0.001); (ii) smokers (23%) have significantly higher adduct levels than non smokers (p < 0.001); iii) non smokers who consume PAH-rich meals > or =52 times/year (142 subjects, 42%) have significantly increased adduct levels than those <52 times/year (p < 0.01). Dietary and smoking habits did not influence the occupationally-induced adduct levels in coke-oven workers. This is the first study that examines anti-B[a]PDE-DNA adduct levels in a large cohort showing that anti-B[a]PDE-DNA adducts can be detected in humans environmentally exposed to low doses of PAH (B[a]P and are modulated by smoke and dietary habits.

[Individual susceptibility to occupational carcinogens: the evidence from biomonitoring and molecular epidemiology studies]. / Suscettibilità individuale ai cancerogeni occupazionali: evidenze dal monitoraggio biologico e dall'epidemiologia molecolare.

This paper reviews the literature on the influence of metabolic and DNA repair polymorphisms of biological indicators of genotoxic risk commonly used in biomonitoring occupational exposure to carcinogens. Genetic polymorphisms which influence biomarkers (urinary metabolites, protein and DNA adducts), include P450 cytochromes (CYPs) and glutathione S-transferases (GSTs) in exposure to polycyclic aromatic hydrocarbons (PAHs), and acetyltransferases (NATs) in exposure to aromatic amines (AAs). As regards exposure to benzene, also relevant is the influence of epoxydohydrolase (EPHX) and NAD(P)H quinone oxidoreductase (NQO1) on the urinary excretion of t,t-muconic and phenylmercapturic acids. With respect to occupational exposure to styrene, EPHX significantly influences the levels of Chromosome Aberrations (CAs), strongly predictive genotoxic biomarkers of cancer risk. Some recent studies examine the role of polymorphisms linked to DNA repair genes in the modulation of genotoxic risk associated with PAH exposure, both for life-style (dietary and smoking behaviour) and for occupational reasons. In addition, molecular epidemiology studies (case/control studies) of lung cancer in smokers published since 2000 may also be viewed as representing models of effects due to exposure to carcinogenic mixtures, some of which are present in the working environment (e.g., BaP, benzene, AAs). Almost all studies show the clearcut influence (i.e., increased lung cancer risk with OR > or = 2) of genetic polymorphisms linked to PAH metabolism (in particular, CYPIA1, GSTM1 and P1). Among the risk factors are the different mutagen sensitivity towards, for instance, bleomycin and BaP (tested in vitro), the reduced repair capacity to DNA damage induced by BaP, and increases in some biomarkers of early biological effect (DNA adducts and stable CAs). Other risk factors, such as heredity (siblings of cancer patients have a risk factor > or = 3 with respect to the general population), ethnicity (Chileans > Caucasians; Japanese > Americans) and gender (women > men), have still not been clearly characterized and these are also reported in this paper. It is clear from the above that genetic differences underlie individual susceptibility to lung cancer, whether caused by exposure to tobacco smoke or to occupational carcinogens like PAHs. Some of these indicators of exposure/individual susceptibility can be evaluated in groups at high risk of occupational lung cancer, such as coke-oven and aluminium workers and those exposed to coal tar fumes and soot, etc., with the aim of identifying subjects who are susceptible due to the high concentrations of carcinogens found in their working environment.

Non-smoking coke oven workers show an occupational PAH exposure-related increase in urinary mutagens.

We examined the urinary mutagenicity in the YG1024 Salmonella typhimurium strain in the presence of S9 mix, of 31 male non-smoking coke oven workers and an equal number of controls matched for gender and dietary habits. Occupational PAH exposure to the workers was assessed by means of the individual urinary post-shift excretion of 1-pyrenol (mean +/- S.D.: 5.41 +/- 6.06 micromole/mol creatinine). Eleven urinary extracts of workers (35.5%) were clearly mutagenic (with at least a doubling of the number of spontaneous revertants), against only two samples in the control group (6.5%) (chi2-test; chi2 = 7.883; P < 0.01). Moreover, the mean mutagenic activity level corrected for dilution/concentration of the urine was about three times higher in coke oven workers than in matched controls (mean +/- S.D. (range) 495 +/- 407 (89.7-1603) versus 186 +/- 113 (14.2-524) net revertants/mmol creatinine; Mann-Whitney U-test, z = 3.86, P < 0.001). Simple linear regression analysis showed that the coke workers' urinary mutagenic activity is associated with the PAH occupation-related urinary excretion of 1-pyrenol (r = 0.41, P = 0.0215). This study definitely demonstrates an occupation-related exposure of coke oven workers' bladder epithelium to mutagenic PAH metabolites. This factor, mainly in the case of high exposure studied here, may account for a higher bladder cancer risk in coke oven workers.

[Molecular epidemiology in occupational medicine: methodological features and impact of individual genetic susceptibility]. / Epidemiologia molecolare in medicina del lavoro: aspetti metodologici e influenza della suscettibilità genetica individuale.

A review of main methodological questions regarding biomarkers is reported focusing on validation, laboratory variability, study design and statistical analysis. The indicated perspective is the setup of protocols finalized at the study of multiple panels of genotoxicity biomarkers taking into account the influence of gene-environment interaction at low doses, of the modulation of the biomarkers associated to the genetic polymorphism. An overview on the influence of metabolic and DNA repair polymorphisms on biological indicators of genotoxic risk in occupational, environmental or life-style exposure is also presented. Genetic polymorphisms that influence human genotoxic risk are those of glutathione s-transferase and cytochrome P450 in exposure to polycyclic aromatic hydrocarbons (PAHs), those of N-acetyltransferase in both occupational and environmental exposures to aromatic amines (AAs) and similar compounds. Lastly recent and important studies, on the effect of the newly discovered polymorphisms affecting DNA repair enzymes on the modulation of genotoxic risk linked to life style (i.e., aflatoxin and PAHs from diet) and smoking behaviour and to environmental genotoxic exposure, are reported. To date biomarkers represent a new tool for epidemiological research in occupational medicine and they could represent a valid instrument for group evaluation but they are not useful for the risk assessment on individual basis. To achieve this objective it is necessary to demonstrate a stronger association with the endpoint that perhaps the future development of genetic and molecular epidemiology will make possible.

[CYP1A2, NAT2, and GSTM1 phenotype/genotype modulate human exposure and various environmental mutagens: our experience]. / I feno/genotipi CYP1A2, NAT2 e GSTM1 modulano l'esposizione umana ad alcuni mutageni ambientali: la nostra esperienza.

Since some years in our research group has been studied the influence of metabolic genotypes on two biomarkers of genotoxic risk (BPDE-DNA adducts and urinary mutagenicity) in humans exposed to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines. The aim was to identify possible genetic susceptible factors capable of modulating individual response to these carcinogens. Humans exposed to PAHs: dermatological patients therapeutically treated with coal tar based ointments (CT), coke oven workers and chimney sweeps. People exposed to aromatic amines will be volunteers after a meal of pan-fried hamburgers and smokers. An overview of the results we found until now will be presented.

Polyaromatic hydrocarbons administered in humans by dermal route increase total IgE.

Inhalation of polyaromatic hydrocarbons (PAHs) extracted from diesel exhaust particles (DEP) enhances local (nasal) production of IgE in humans. The aim of the present research is to investigate whether in humans dermal exposure to PAHs which are not extracted from DEPs increases serum IgE, and whether host factors modify the immunologic effect. In thirty-two patients with acute psoriatic lesions, a cream containing 3% of coal tar (which holds a variety of PAHs) was applied to the skin for 24 hours. Serum IgE were measured before (IgE0) and four (IgE4) and eight (IgE8) days after application. Replicated means were compared by analysis of variance for repeated measures and by the Newman-Keuls' test. IgE0, IgE4 and IgE8 were 151.19, 159.69 (a 6% excess) and 170.90 kU/L (a 13% excess) respectively; pairwise comparison showed IgE8 was significantly higher than IgE0 (p<0.05). At multiple linear regression analysis, the percentage increase in serum IgE across observation days was the dependent variable against age, sex, cigarettes/day, urinary 1-pyrenol, atopy, skin area treated, and grams of cream. Of the independent variables, only age had a significant (p<0.028) influence: the younger the age, the higher the IgE response to PAHs. We conclude that whatever the source and the route of entry (skin or respiratory tract), PAHs increase total serum IgE, mainly in younger age groups.

Lung cancer risk in workers exposed to poly(vinyl chloride) dust: a nested case-referent study.

BACKGROUND: There have been few investigations of an association between poly(vinyl chloride) (PVC) dust exposure and an increase in lung cancer incidence, and their conclusions have been inconsistent. AIMS: To determine whether PVC and/or vinyl chloride monomer (VCM) is the associated risk factor(s), by means of a nested case-referent study, in order to estimate lung cancer risk, avoiding selection, information, or confounding biases. METHODS: Thirty eight cases of histologically verified lung cancer and 224 control subjects without a history of cancer were selected from an Italian cohort of 1658 vinyl chloride workers. Information sources included clinical records (diagnosis, smoking habits) and plant records (occupational history). The risk of lung cancer was estimated by odds ratios (OR) with 95% confidence intervals (CI), calculated using logistic regression models. RESULTS: In PVC baggers exposed to high levels of respirable PVC particles in the workplace, the lung cancer OR increases by 20% for each extra year of work (OR = 1.2003; 95% CI 1.0772 to 1.3469; p = 0.0010), when the influence of age and smoking habits is controlled. No relation was found between lung cancer and cumulative VCM exposure. CONCLUSION: This nested case-control study showed, in the VCM/PVC industry, an increased risk of lung cancer associated with exposure to PVC dust; previous cohort studies failed to recognise such excess, probably because they used VCM exposure as the risk indicator.

Role of metabolic polymorphisms NAT2 and CYP1A2 on urinary mutagenicity after a pan-fried hamburger meal.

In this work the phenotyping approach was used to study the influence of metabolic polymorphisms NAT2 and CYP1A2 on S9-mediated urinary mutagenicity, detected with Salmonella strain YG1024, in 50 subjects after a meal of pan-fried hamburgers. All 50 post-meal samples, but not pre-meal ones, were clearly mutagenic (number of urine samples able to double number of spontaneous revertants was 50 to 0, respectively). CYP1A2 positively influences urinary mutagenicity: a rise in CYP1A2 activity increases levels of post-meal urinary mutagens (1.16+/-0.91 vs 1.72+/-1.19 7-h minimum mutagenic doses (MMDs)/intake), especially in NAT2 slow acetylators (2.18+/-1.33 vs 0.90+/-0.54 7-h MMDs/intake, Mann-Whitney U-test, P<0.05). NAT2 rapid acetylators exert lower post-meal urinary mutagenicity than slow ones (1.41+/-1.02 vs 1.77+/-2.45 7-h MMDs/intake) and even more if the latter are extensive CYP1A2 metabolizers (1.41+/-1.02 vs 2.18+/-1.33 7-h MMDs/intake), but the difference did not reach statistical significance. In conclusion, this study indicates that CYP1A2 and NAT2 activities influence the presence of urinary mutagens after a meal of pan-fried hamburger (rich in HHAs) and consequently their potential genotoxic risk.

Environmental and biological monitoring of traffic wardens from the city of Rome.

A molecular epidemiological study on Roman policemen is ongoing. The results of a first assessment of the occupational exposure to aromatic compounds of 66 subjects engaged in traffic control and of 33 office workers are presented in this paper. Passive personal samplers and urinary biomarkers were used to assess exposure to benzene and polycyclic hydrocarbons during work shifts. The results obtained indicate that benzene exposure in outdoor workers is about twice as high as in office workers (geometric mean 7.5 and 3.4 micrograms/m3, respectively). The distribution of individual exposure values was asymmetrical and skewed toward higher values, especially among traffic wardens. Environmental benzene levels recorded by municipal monitoring stations during work shifts (geometric mean 11.2 micrograms/m3) were in the first instance comparable to or greater than individual exposure values. However, several outlier values were observed among personal data that greatly exceeded average environmental benzene concentrations. Among the exposure biomarkers investigated, only blood benzene correlated to some extent with previous exposure to benzene, while a seasonal variation in the excretion of 1-hydroxypyrene and trans-muconic acid was observed in both study groups. In conclusion, these results suggest that outdoor work gives a greater contribution than indoor activities to benzene exposure of Roman citizens. Moreover, relatively high-level exposures can be experienced by outdoor workers, even in the absence of large-scale pollution episodes.

Biological indicators of genotoxic risk and metabolic polymorphisms.

International scientific publications on the influence of metabolic genotypes on biological indicators of genotoxic risk in environmental or occupational exposure are reviewed. Biomarkers of exposure (substance or its metabolites in biological fluids, urinary mutagenicity, protein and DNA adducts) and of effects (chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (Mn), COMET assay, HPRT mutants) have been evaluated according to different genotypes (or phenotypes) of several activating/detoxifying metabolic activities. In less than half the studies (43 out of 95), the influence of genotype on the examined biological indicator was found, of which four report poorly reliable results (i.e., with scarce biological plausibility, because of the inconsistency of modulated effect with the type of enzymatic activity expressed). As regards urinary metabolites, the excretion of mercapturic acids (MA) is greater in subjects with high GST activity, that of 1-pyrenol and other PAH metabolites turns out to be significantly influenced by genotypes CYP1A1 or GSTM1 null, and that of exposure indicators to aromatic amines (AA) (acetylated and non-acetylated metabolites) is modulated by NAT2. In benzene exposure, preliminary results suggest an increase in urinary t, t-muconic acid (t,t-MA) in subjects with some genotypes. On urinary mutagenicity of PAH-exposed subjects, the effects of genotype GSTM1 null, alone or combined with NAT2 slow are reported. When DNA adduct levels are clearly increased in PAH-exposed group (18 out of 22), 7 out of 18 studies report the influence of GSTM1 null on this biomarker, and of the five studies which also examined genotype CYP1A1, four report the influence of genotype CYP1A1, alone or in combination with GSTM1 null. A total of 25 out of 41 publications (61%) evaluating the influence of metabolic polymorphisms on biomarkers of effect (cytogenetic markers, COMET assay, HPRT mutants) do not record any increase in the indicator due to exposure to the genotoxic agents studied, confirming the scarce sensitivity of these indicators (mainly HPRT mutants, Mn, COMET assay) for assessing environmental or occupational exposure to genotoxic substances. Concluding, in determining urinary metabolites for monitoring exposure to genotoxic substances, there is sufficient evidence that genetically-based metabolic polymorphisms must be taken into account in the future. The unfavourable association for the activating/detoxifying metabolism of PAH is also confirmed as a risk factor due to the formation of PAH-DNA adducts. The clearly protective role played by GSTT1 on DEB (and/or related compound)-induced sister chromatid exchanges (SCEs) should be noted. The modulating effects of genotypes on protein adduct levels in environmental and occupational exposure have not yet been documented, and most studies on the influence of genotype on biological indicators of early genotoxic effects report negative results.

[Assessment of occupational exposure to aromatic polycyclic hydrocarbons determining urinary levels of 1-pyrenol]. / Valutazione dell'esposizione professionale ad idrocarburi policiclici aromatici mediante l'analisi dei livelli urinari di 1-pirenolo.

In conformity with Italian law 626/94, occupational exposure to Polycyclic Aromatic Hydrocarbons (PAH) in several types of work environments was assessed by analysing urinary levels of 1-pyrenol. A total of 231 non-smokers exposed to PAH (82 workers, employed in two different thermoelectric power plants using combustible oil (30 subjects from plant A and 52 from plant B), 18 subjects working for a company recovering exhausted oils, 12 working on rubber production, 10 on road surface asphalting operations, 22 working in the anodizing section of an aluminium plant, 27 chimney-sweeps, and 60 coke-oven workers (30 topside workers, and 30 doing other jobs)) were enrolled. There were also 53 non-smoker control subjects, not occupationally exposed to PAH. Current smokers were excluded, since smoking is an important confounding factor when occupational exposure to low PAH concentrations are monitored. Confounding factors, i.e., diet and passive smoking, were checked by means of a questionnaire which, in addition to personal data and habits, also requested specific details about the type of diet followed and possible exposure to passive smoking during the 24-hour period preceding urine collection. In controls, exposure to PAH in the diet significantly increased 1-pyrenol levels in urine: in subjects introducing > or = 1 microgram of pyrene with the diet, the mean urinary level of 1-pyrenol was significantly higher than that introduced with < 1 microgram (high versus low dietary intake, mean +/- SD, 0.08 +/- 0.13 and 0.04 +/- 0.06 1-pyrenol mumoles/mole of creatinine, respectively; Mann-Whitney U-test Z = 2.67, p < 0.01). Conversely, passive smoking did not influence 1-pyrenol levels. In the overall population (controls and exposed), multiple linear regression analysis showed that levels of urinary 1-pyrenol were significantly influenced by occupational exposure to PAH in asphalt workers, anodizing plant workers, chimney-sweeps, and coke-oven workers, both those working at the top side of the oven and those doing other jobs (t = 2.19, p = 0.02; t = 2.56, p = 0.01; t = 5.25, p = 0.001; t = 3.34, p = 0.001; t = 7.82, p = 0.001, respectively; F = 9.7, p < 0.01), but not in power plant workers in contact with combustible oils, workers recovering exhausted oils, or rubber production workers. Diet and passive smoking did not influence urinary 1-pyrenol levels in the entire sample population. This biomarker also allowed an assessment of exposure levels among certainly exposed subjects. The percentage of subjects with urinary 1-pyrenol values higher than the 99th percentile of the reference population (0.67 mumoles 1-pyrenol/mole of creatinine) was significantly higher than that of controls in asphalt workers (20%), anodizing plant workers (14%), chimney-sweeps (13%) and coke-oven workers (33%) (chi-square test: asphalt workers = 6.1, p = 0.01; anodizing plant workers = 4.3, p = 0.04; chimney-sweeps = 7.1, p = 0.008; coke-oven workers with other duties = 4.4, p = 0.04; top side workers = 16.5, p < 0.001). In chimney sweeps and top side workers, respectively 2 and 4 subjects (7% and 13%) exceeded the precautionary level of 1.4 mumoles 1-pyrenol/mole of creatinine; of these, 1 chimney sweep and 3 top side workers (4% and 10%) exceeded the recommended biological threshold of 2.3 mumoles 1-pyrenol/mole of creatinine.

[Biomarkers of gentotoxic risk and metabolic polymorphism]. / Indicatori biologici di rischio genotossico e polimorfismi metabolici.

This paper reviews studies published in the international scientific literature evaluating the influence of genetically based metabolic polymorphisms on biological indicators of genotoxic risk in environmental or occupational exposure. Exposures due to life style (i.e. diet or smoking) were not considered. Indicators are subdivided into internal dose indicators (concentration of the substance or its metabolites in biological fluids, urinary mutagenicity, adducts of hemoglobin, plasma proteins and DNA), and early biological effects (chromosome aberrations, sister chromatid exchanges, micronuclei, COMET assay, HPRT mutants). The metabolic genotypes (or phenotypes) examined by various authors are: ALDH2 (aldehyde dehydrogenase), CYP (P450 cytochrome) 1AI, CYP1A2, CYP2E1, CYP2D6, EPHX (epoxidohydrolase), NAT2 (N-acetyl transferase), NQO1 (NAD(P)H: kinone oxidoreductase), PON1 (paraoxonase), GST (glutathione S-transferase) M1, GSTT1 and GSTP1. In more than half the studies (52 out of 96), no influence of genotype was found in the biological indicator. This may be due either to the poor sensitivity of the indicator used, or to low exposure. In studies examining the effect of genotype on the indicator, the biological plausibility of the result was evaluated, i.e., whether the effect is consistent with the type of enzymatic activity expressed. Four studies reported not very reliable results and suggest either the unfavourable influence of genotype GSTM1 with high detoxifying activity, or enzymatic activity poorly involved in the metabolism of the xenobiotics in question (NAT2 in the case of PAH). As regards urinary metabolites of genotoxic agents, eight studies reported the modulating effect of genotype. The urinary excretion of mercapturic acids was greater in subjects with high GST activity. In exposure to PAH, urinary 1-pyrenol and PAH metabolites turn out to be significantly influenced by genotypes CYP1A1 or GSTM1 null; in exposure to aromatic amines, the influence of NAT2 on exposure indicators (levels of acetylated and non-acetylated metabolites) was confirmed. Exposure to benzene led to an increase in t-t-MA in some genotypes, although experimental verification is still necessary. As regards urinary mutagenicity, the effect of genotype GSTM1 null is reported, and of the same genotype combined with NAT2 slow, in non-smoking individuals subjected to high exposure to PAH and in cigarette-smoking/coke-oven workers. Lastly, the determination of urinary metabolites in monitoring exposure to genotoxic substances, provides sufficient evidence that genetically based metabolic polymorphisms must be taken into account in the future. There is still little evidence regarding the importance of genotype on the level of protein adducts in environmental and occupational exposure. A relatively large number of publications (22) dealt with DNA adduct levels in PAH exposure. In 18 studies, the biological indicator clearly increases with respect to values in control subjects. Of these studies, seven reported the influence of GSTM1 null on DNA adducts and, of the five studies which also examined genotype CYP1A1, four reported the influence on DNA adduct level of genotype CYP1A1, alone or in combination with GSTM1 null. It therefore seems as if the unfavourable association for the activating/detoxifying metabolism of PAH is a risk factor for the formation of PAH-DNA adducts. Most publications (25 out of 41; 61%) dealing with metabolic polymorphisms in effect indicators (cytogenetic markers, COMET assay, HPRT mutants) did not report any increase in the indicator due to exposure to the genotoxic agents studied. These indicators of genotoxic damage, including mainly the frequency of HPRT mutants (100%), Mn (90%) and the COMET assay (67%), are not sufficiently sensitive in revealing exposure, confirming that they are not particularly suitable for measuring exposure to genotoxic substances in occupational or environmental exposures. It is therefore difficult to assess the influence of metabolic genotypes by means of this type of biological indicator. The few positive results reported for SCE in occupational studies mentioned the influence of genotype ALDH2, either alone or in combination with genotype CYP2E1 in exposure to CVM, or in combination with GSTM1 null in exposure to epichlorohydrin. For CA the results showed unfavourable combinations of genotypes CYP2E1, GSTM1 and PON1 in exposure to pesticides, and GSTM1 null in combination with NAT2 slow in exposure to urban air. All the remaining studies on the effect of genotype on biological indicators of cytogenetic damage reported negative results.

Influence of GSTM1 genotypes on anti-BPDE-DNA adduct levels in mononuclear white blood cells of humans exposed to PAH.

OBJECTIVE: Association between genetic deletion polymorphism of GSTM1 (*0/*0 or active) and levels of anti (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)-DNA adducts in the peripheral blood lymphocyte plus monocyte fraction (LMF) of PAH-exposed subjects was investigated. METHODS: A total of 94 Caucasian subjects comprised the sample population: 13 coke-oven workers, 19 chimney sweeps, 36 aluminum-anode plant workers, and 26 non-occupationally PAH-exposed subjects (controls). PAH exposure was assessed in each group by means of the urinary excretion of 1-pyrenol (mean group levels 1.2, 0.7, 0.3, and 0.1 mumol/mol creatinine in coke-oven workers, chimney sweeps, aluminum-anode plant workers, and control subjects, respectively). Anti-BPDE-DNA adducts were detected by HPLC/fluorescence analysis of anti-BPDE tetrols (tetrol I-1) released after acid hydrolysis of DNA samples. RESULTS: In coke-oven workers the percentage of cases with adduct levels exceeding the 95th percentile control value (4.4 adducts/10(8) nucleotides) was significantly higher in the subgroup with the null GSTM1 genotype (*0/*0) (100%) than in that with active GSTM1 (43%; chi 2 test, P < 0.05). In the other groups with different and lower levels of PAH exposure the percentages of positive samples were always higher in the subgroup with GSTM1 *0/*0 than in the active one, although the differences were not statistically significant. Univariate (odds ratio) and multivariate (relative risk) analyses showed that the risk of having high anti-BPDE-DNA levels increased with occupational exposure to PAH. Such risks, moreover, were further significantly increased by the lack of GSTM1 activity (RR = 5.94; CI = 1.15-30.7; P < 0.05). In coke-oven workers, chimney sweeps, and aluminum workers, respectively, the multiplicative effect of the null genotype with occupational PAH exposure gives risks of 162 (= 27.2 x 5.94), 10 (= 1.70 x 5.94), and 3 (= 0.50 x 5.94) times higher probability (risk) of high BPDE-DNA adduct formation than that of non-exposed subjects with the active GSTM1 genotype. CONCLUSION: Our results indicate a greater risk of anti-BPDE-DNA adduct formation resulting from occupational high-level PAH-exposure in GSTM1 null (GSTM1 *0/*0) workers.

HPLC/fluorescence determination of anti-BPDE-DNA adducts in mononuclear white blood cells from PAH-exposed humans.

The aim of this study was to compare (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)-DNA adduct levels in groups of humans subjected to various levels of polycyclic aromatic hydrocarbon (PAH) (benzo[a]pyrene) exposure. An HPLC/fluorescence method was applied to detect specifically anti-BPDE-DNA adducts in mononuclear white blood cells [lymphocyte plus monocyte fraction (LMF)] from humans exposed to PAHs. A total of 130 subjects comprised the sample population: 26 psoriatic patients (3 days after clinical coal tar treatment of the skin), 15 coke oven workers, 19 chimney sweeps, 36 aluminium anode plant workers and 34 non-occupationally PAH-exposed subjects (controls). PAH exposure was assessed in each group by means of the urinary excretion of 1-pyrenol (mean group levels: 1.2, 0.7, 0.3, 65.0 and 0.1 micromol/mol creatinine in coke oven workers, chimney sweeps, aluminium plant anode workers, psoriatic patients and non-occupationally PAH-exposed subjects, respectively). HPLC/fluorescence analysis of BPDE-DNA adducts showed that the percentage of subjects with adduct levels exceeding the 95 percentile control subject value (8.9 adducts/10(8) nucleotides) was significantly high in coke oven workers (46.7%) and chimney sweeps (21.0%) (chi2 test, P < 0.01 and P < 0.05, respectively) but not in aluminium plant workers (11.1%) and psoriatic patients (0%). The increase in BPDE-DNA adduct levels in LMF (Ln values) was significantly related to chronic inhalatory and high PAH exposure (linear multiple regression analysis, F = 6.37, P < 0.01; t = 4.2, P < 0.001). Skin acute (or short-term) and high PAH exposure, charcoal-grilled meat consumption and smoking habit did not seem to influence BPDE-DNA adduct formation in LMF.

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar.

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467. 8+/-211.0 nmoles-24 h (range 94.6-890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693+/-30 867 vs. 6877+/-6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0. 6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null (n=6) were on average higher than those of GSTM1-positive subjects (n=9) (55 498+/-45 957 vs. 34 156+/-11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136. 8+/-34.7 vs. 70.8+/-23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann-Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.

Urinary mutagenicity on TA98 and YG1024 Salmonella typhimurium strains after a hamburger meal: influence of GSTM1 and NAT2 genotypes.

Mutagenicity on TA98 and YG1024 Salmonella typhimurium strains of pan-fried hamburger extracts and of 24 h post-meal urine from 32 non-smoking volunteers was evaluated. Each participant in the study was GSTM1 and NAT2 genotyped. After cooking the meat showed mutagenic activity (mean +/- SD) on strains TA98 and YG1024 of 114 +/- 129 and 1437 +/- 1536 net revertants/g respectively. Twenty three of 32 urine samples showed clear mutagenic activity (i.e. caused at least a doubling of the number of spontaneous revertants) on the O-acetyltransferase over-producing strain YG1024, while none of the post-meal 24 h urine samples was clearly mutagenic on strain TA98. Total 24 h post-meal YG1024-active urinary mutagens were well correlated with the levels of mutagen intake with the meal (r2 = 0.5977, F = 44.58, P < 0.01). In the group under study GSTM1 genotypes did not influence urinary mutagenicity. Highly exposed subjects (n = 15) with the NAT2-ss genotype showed significantly increased levels of urinary mutagenicity on strain YG1024 in comparison with NAT2-R subjects (mutagen intake-adjusted total 24 h mutagen excretion = 1.00 +/- 0.29 versus 0.66 +/- 0.32, Mann-Whitney U test, U = 12.5, P < 0.05). Our results suggest that the levels of urinary mutagens derived from diets rich in heterocyclic aromatic amines, which are specifically detected by the YG1024 Salmonella strain, are modulated by NAT2-dependent enzyme activity, slow acetylators having higher levels of mutagens in their urine. Subjects with the rapid acetylator genotype, who are known to be at risk for colon cancer, seem to be partially protected with respect to the risk of bladder cancer.

Analysis and clinical implications of p53 gene mutations and human papillomavirus type 16 and 18 infection in primary adenocarcinoma of the uterine cervix.

Mutant p53 is frequently detected in endometrial and ovarian carcinoma, but it is rare in cervical cancers. Previous reports focused on cervical squamous cell carcinoma, whereas cervical adenocarcinoma was given little attention. We searched for p53 gene mutations in 74 primary cervical adenocarcinomas with known human papillomavirus (HPV) status. Our aim was to evaluate the prevalence of p53 mutations and to investigate their possible role as an independent prognostic factor. We found mutations in 13.5% with a high rate of G:C --> A:T transitions as observed in endometrial adenocarcinoma. As p53 mutations are more frequently detected in malignancies of high grade, high stage, and large size, this molecular event seems to play a role in the progression rather than in the induction of cervical adenocarcinoma. In our series, patients with HPV-negative tumors and patients with mutated neoplasms, irrespective of HPV infection, had a shorter survival. Yet the absence of HPV infection and presence of p53 mutations are not independent risk factors for tumor-related death after adjustment for clinicopathological confounders. The only significant and independent predictors of survival are age of patient, stage of disease, tumor grade, and presence of lymph node metastases.