The usefulness of three biallelic restriction fragment length polymorphisms versus a polymorphic dinucleotide tandem repeat polymorphism at the low-density-lipoprotein receptor gene locus for diagnosis of familial hypercholesterolemia

The DNA adduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been widely used as a biomarker for oxidative stress. Bulky DNA adducts, which are detectable by the 32P-postlabelling method, provide evidence for exposure to and metabolic activation of large, mainly apolar compounds, e.g. polycyclic aromatic hydrocarbons. We determined both types of adducts in placental tissues of 30 term pregnancies and related the adduct levels to the exposure to tobacco smoke and the plasma antioxidant status. Urine and plasma continine concentrations were used to select 10 nonsmokers, 9 nonsmokers exposed to environmental tobacco smoke (ETS) and 11 smoking women. Placental levels of 8-OHdG were 0.84 +/- 0.11, 0.90 +/- 0.21 and 0.83 +/- 0.20/10(5) deoxyguanosine bases (dG) for nonsmokers, nonsmokers exposed to ETS and smokers, respectively. The differences between the groups were not significant. Smoking women had significantly lower plasma vitamin C and beta-carotene concentrations than nonsmoking women or nonsmoking women exposed to environmental tobacco smoke. The 8-OHdG adduct level in placental DNA was inversely correlated with the plasma vitamin E concentration (r = -0.47, P < 0.05). There was no association between placental 8-OHdG adducts and vitamin A, C and beta-carotene in plasma. In total, 15 different adducts could be identified in the 30 placenta samples by the 32P-postlabelling method. There was a strong inter-individual variation in both the number of adducts and adduct intensities. No smoking-related or vitamin-related effects on adduct patterns or intensities were found. Our findings suggests that, within the limits of the methods used, tobacco smoke exposure during pregnancy does not lead to a measurable increase in placental DNA adduct levels and that vitamin E appears to have a protective effect on placental 8-OHdG formation.     

Influence of feed restriction in primiparous lactating sows on body condition and metabolic parameters

Bearing in mind a great diffusion of electromagnetic fields (EMF) with power-line frequency (in Poland-50 Hz) both in the occupational and communal environments, it is not surprising that possible health effects related to this exposure evoke much interest. Electromagnetic fields may affect the circulatory and nervous systems because of theoretical probability that electric impulses, generated by external electric and magnetic fields, may disturb their functions. For this reason we have decided to evaluate the functioning of the circulatory system in persons occupationally exposed to power-line frequency electromagnetic fields by employing the most up-to-date methods facilitating the in-depth diagnosis of the circulatory system and neurovegetative mechanisms. The work presented focused on the evaluation of electrocardiographic changes. The study covered 63 workers of the transforming and distributing stations, aged 22-67 years (median 39 +/- 10), employed under exposure for 2-43 years (median 15 +/- 10). The control group consisted of 42 workers of radio link stations, aged 23-65 years (median 30 +/- 14), employed in the similar system but not exposed to EMF, with employment duration of 1-42 years (median 13 +/- 4). All persons were subject to general medical examinations, resting ECG, and 24 h Holter monitoring. In addition, the level of exposure in individual workplaces was estimated following the measurements of the intensity of electric and magnetic fields. In workers of electromagnetic stations an increased risk for electrocardiographic disturbances was revealed. Under conditions of exposure to electric fields, observed in stations where workers were employed, the risk was increased by 10%.     

Oxidative DNA damage estimated by oxo8dG in the liver of guinea-pigs supplemented with graded dietary doses of ascorbic acid and alpha-tocopherol

Dietary antioxidants may influence cancer risk and aging by modifying oxidative damage. The effect of graded dietary doses of the antioxidant vitamins C and E on oxidative DNA damage was studied in the liver of guinea-pigs under normal conditions. Like human beings, guinea-pigs cannot synthesize ascorbate and alpha-tocopherol. In one experiment, three groups of 6-8 guinea-pigs were fed diets containing 15 mg of vitamin E/kg chow and three different amounts of vitamin C (33,660 or 13,200 mg/kg) for 5 weeks. In a second experiment, three groups of seven guinea-pigs were fed diets containing 660 mg of vitamin C/kg and three different amounts of vitamin E (15, 150 or 1500 mg/kg) for 5 weeks. The three graded levels of each vitamin respectively represent marginal deficiency, an optimum supplementation and a megadose. Oxidative damage to liver DNA was estimated by measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG) referred to deoxyguanosine (dG) by means of high-performance liquid chromatography with simultaneous electrochemical-coulometric and ultraviolet detection. The level of ascorbate in the liver was 0.034 +/- 0.051, 1.63 +/- 1.06 and 1.99 +/- 0.44 micromol/g in the low, medium and high dose ascorbate groups (59-fold variation). The liver concentration of alpha-tocopherol was 28 +/- 11, 63 +/- 18 and 187 +/- 34 nmol/g in the low, medium and high dose alpha-tocopherol groups (7-fold variation). The level of oxo8dG in the liver DNA was 1.89 +/- 0.32, 1.94 +/- 0.78 and 1.93 +/- 0.65 per 10(5) dG in the low, medium and high dose ascorbate groups (no effect: P > 0.05). In the low, medium and high dose alpha-tocopherol groups oxo8dG level in the liver DNA was 2.85 +/- 0.70, 2.74 +/- 0.66 and 2.61 +/- 0.92 per 10(5) dG (no effect: P > 0.05). It is concluded that even very large variations in the content of the antioxidant vitamins C and E in the diet and liver have no influence on the steady-state level of oxidative damage to guanine in the liver DNA of normal unstressed guinea-pigs.     

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p-XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.     

Neurophysiological findings among workers occupationally exposed to styrene

Ninety-six male workers occupationally exposured to styrene in 24 plants producing reinforced polyester plastic products were studied neurophysiologically. The mean age of the subjects was 29.6 +/- 7.4 years, and the duration of the exposure varied from 6 months to 14 years (mean 5.0 years). Mandelic acid concentration in the urine (mean of five values determined during five consecutive weeks) was used as the measure of exposure. The individual means of the mandelic acid concentrations varied from 7 to 4,715 mg/dm3 (median 808 mg/dm3). Neurotoxic effects of styrene exposure upon the central nervous system were revealed by abnormalities in the EEG. The overall prevalence of abnormal EEGs was 24% (23 out of 96), which is higher than that found among a normal population (p less than 0.05). Abnormal EEGs were found in one-third of the subjects with relevant styrene exposure (mandelic acid concentration over 700 mg/dm3), while low-level styrene exposure did not increase the prevalence of normal EEGs above that found in a normal population. Nerve conduction velocity measurements performed on 40 subjects did not reveal any definite relationship between neuropathy and styrene exposure. The EEG examination is useful in the investigation of the neurotoxic effects of styrene. Whenever a possibility exists of excessive styrene exposure or beginning poisoning, the workers should be submitted to an EEG examination.     

Formation of 8-hydroxydeoxyguanosine from 2'-deoxyguanosine and its decomposition by active oxygen

8-hydroxydeoxyguanosine (8-OHdG) was formed from dG and isolated DNA by photosensitization with rose bengal (RB) and methylene blue (MB). 8-OHdG formed from dG was decomposed by the photosensitization with these dyes. Singlet oxygen was concerned with the formation and decomposition of 8-OHdG by photosensitization. Fe++ oxidized dG to 8-OHdG, which was decomposed by the addition of H2O2. 8-OHdG was formed and decomposed by the treatment of dG with Fe++, EDTA and ascorbic acid. Hydroxy radical (.OH) participated in the formation and decomposition of 8-OHdG by Fe++.     

Induction of 8-hydroxydeoxyguanosine in isolated DNA and HeLa cells exposed to fecapentaene-12: evidence for the involvement of prostaglandin H synthase and iron

The genotoxic/mutagenic mechanism(s) of action of fecapentaene-12 (fec-12) is complex but there is evidence to suggest that the generation of active oxygen species (AOS) may be involved. This has been assessed by measuring the formation of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA and HeLa cells exposed in vitro to fec-12. The possibility that fec-12 may form AOS via peroxidative 'activation' by prostaglandin H synthase (PHS) has been investigated by measuring 8-OHdG in HeLa cells exposed to fec-12 in the absence or presence of PHS inhibitors. The role of iron as a catalyst in this pathway has also been investigated. A 4-fold increase in the level of 8-OHdG in isolated DNA was seen after exposure to fec-12 (1 mM) alone. This increase was enhanced synergistically by ferrous iron. Fec-12 exposure of HeLa cells at 50 and 100 microM induced 2- and 3-fold increases (P < 0.001) respectively in the level of 8-OHdG in cellular DNA. No increase was seen at 10 microM fec-12. The PHS inhibitors indomethacin and acetylsalicylate blocked the formation of 8-OHdG induced by fec-12 (50 microM) but did not inhibit the formation of 8-OHdG in these cells after exposure to H2O2 and Fe2+. Addition of the iron chelating agent o-phenanthroline to cells prior to fec-12 exposure blocked the increase in 8-OHdG induced by fec-12 (50 microM). Addition of the radical scavenging agent DMSO (10%) to cells prior to fec-12 exposure reduced the level of 8-OHdG to within 10% of control. Specific inhibition of fec-12 induced 8-OHdG formation in HeLa cells by PHS inhibitors suggests that this enzyme may be involved in 'activating' fec-12 to form AOS in cells. Inhibition of fec-12 induced 8-OHdG formation in cells by o-phenanthroline suggests a role for intracellular iron as a catalyst in this process.     

Increased DNA oxidation and decreased levels of repair products in Alzheimer's disease ventricular CSF

One of the leading etiologic hypotheses regarding Alzheimer's disease (AD) is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Although several recent studies show an increase in levels of brain DNA oxidation in both aging and AD, there have been no studies of levels of markers of DNA oxidation in ventricular CSF. This is a study of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, in intact DNA and as the "free" repair product that results from repair mechanisms. Free 8-OHdG was isolated from CSF from nine AD and five age-matched control subjects using solid-phase extraction columns and measured using gas chromatography/mass spectrometry with selective ion monitoring. Intact DNA was isolated from the same samples and the levels of 8-OHdG determined in the intact structures. Quantification of results was carried out using stable isotope-labeled 8-OHdG. By using this sensitive methodology, statistically significant elevations (p < 0.05) of 8-OHdG were observed in intact DNA in AD subjects compared with age-matched control subjects. In contrast, levels of free 8-OHdG, removed via repair mechanisms, were depleted significantly in AD samples (p < 0.05). Our results demonstrate an increase in unrepaired oxygen radical-mediated damage in AD DNA as evidenced by the increased presence of 8-OHdG in intact DNA and decreased concentrations of the free repair product. These data suggest that the brain in AD may be subject to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for removal of oxidized bases.     

Cis-urocanic acid attenuates histamine receptor-mediated activation of adenylate cyclase and increase in intracellular Ca2+

It has been assumed that oxidative damage, including formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts in kidney DNA due to potassium bromate (KBrO3), a renal carcinogen to both sexes of rats, is involved in its mechanisms of tumor induction. However, despite the presumed existence of a repair enzyme(s) for 8-OHdG, there have been no reports demonstrating the changes in adduct levels during medium- or long-term exposure. To elucidate the actual kinetics regarding this parameter during the early stages of KBrO3 carcinogenesis, we measured 8-OHdG levels in kidney DNA together with cell proliferation in renal tubules in both sexes of rats receiving KBrO3 at a dose of 500 ppm in the drinking water for 1, 2, 3, 4, and 13 weeks. Rapid elevation of 8-OHdG levels was noted in treated male rats which persisted until the end of the experiment. Increased cell proliferation in the proximal convoluted tubules was also observed throughout the experimental period, concomitant with alpha2mu-globulin accumulation. Increase in 8-OHdG levels in treated females first became apparent 3 weeks after the start of exposure, with cell proliferation only elevated at the 13-week time point. The present study, employing the same route and dose of KBrO3 known to cause tumors, strongly suggested the requirement of persistent increase of 8-OHdG for neoplastic conversion. Moreover, a clear sex difference in susceptibility to generation of oxidative stress in kidney DNA was found, in addition to alpha2mu-globulin-dependent variation in cell proliferation in the renal tubules.     

Competitive reactions of peroxynitrite with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG): relevance to the formation of 8-oxodG in DNA exposed to peroxynitrite

We have examined the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in reactions of peroxynitrite with 2'-deoxyguanosine (dG) and calf-thymus DNA. Peroxynitrite reacts with dG at neutral pH, but this reaction does not result in the buildup of 8-oxodG. We also do not find any evidence for the formation of 8-oxodG in calf-thymus DNA upon exposure to peroxynitrite. When 8-oxodG is mixed with 1000-fold excess dG and then allowed to react with peroxynitrite, about 50% of the 8-oxodG is destroyed. The preferential reaction of 8-oxodG is also evident when dG in calf-thymus DNA is partially oxidized in an Udenfriend system and then allowed to react with peroxynitrite. We suggest that 8-oxodG is not produced in peroxynitrite-mediated oxidations of dG and DNA or that it is produced but then is rapidly consumed in further reactions with peroxynitrite. Oxidized DNA bases frequently can be more oxidation sensitive than their corresponding progenitors and, therefore, may be present at] low steady-state concentrations and not represent stable markers of oxidative stress status. The importance of the 8-oxodG/peroxynitrite reaction is discussed in relation to the formation of more stable, secondary oxidation products that might be more useful markers of DNA damage.     

Estimation of reference values for urinary 1-hydroxypyrene and alpha-naphthol in Danish workers

In order to assess environmentally and occupationally related exposures to PAH compounds it is essential to have reference or normal values in human body fluids. The establishment of reliable reference intervals is an absolute pre-requisite in determining relationships between internal PAH exposure in humans and health effects in occupationally exposed workers. In this context the estimation of the biological level of PAH metabolites in urine from reference populations has become increasingly important in the field of environmental and occupational toxicology. The present study describes the calculation of tentative reference values for urinary 1-hydroxypyrene on the basis of two reference populations and for urinary alpha-naphthol on the basis of one reference population in accordance with IFCC recommendations. The study subjects were 115 healthy male workers occupationally exposed to PAH at low levels and 121 reference subjects non-occupationally exposed to PAH. Tentative reference values for urinary 1-hydroxypyrene were estimated. In addition, 236 healthy male workers were used to estimate tentative reference values for urinary alpha-naphthol. The reference populations were described by distribution free one-sided tolerance intervals. The 95% one-sided tolerance limit calculated for 1-hydroxypyrene in urine was 0.053 mumol/mol creatinine for non-occupationally exposed individuals and 0.169 mumol/mol creatinine for low level PAH exposed workers, with the coverage interval (95 +/- 4.5) percent at a probability of 0.95. Thus, the probability was 0.975 that the tolerance interval included at least 90.5% of the distribution. In addition, the probability was 0.025 that the tolerance interval included > 99.5% of the population. The tolerance interval for alpha-naphthol in urine was 5.665 mumol/mol creatinine with the coverage interval (95 +/- 4.5) percent at a probability of 0.95.     

Design, construction, implementation, and cost of a hospital pharmacy cleanroom

The authors sought to clarify in a cross-sectional study the possible associations between homeostatic regulators of calcium and occupational exposure to lead. Subjects were 146 industrial male employees, 56 with and 90 without occupational lead exposure. The main outcome measures were serum concentration of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (calcitriol). The median values of blood lead were 40.5 microg/dl in the exposed group and 4.0 microg/dl in the controls. There were no differences between groups in dietary history and serum calcium levels. PTH and calcitriol levels were significantly higher in the exposed than in the nonexposed subjects (42.0+/-24.2 vs. 33.6+/-14.9 pg/ml, p <0.05; and 83.8+/-27.0 vs. 67.9+/-17.6 pmol/liter, p <0.001, respectively). Multivariate analyses showed that after controlling for possible confounders, occupational lead exposure (no/yes) was independently associated with PTH level (pg/ml) (beta = 7.81, 95% confidence interval (CI) 3.7-11.5) and with calcitriol (pmol/liter) (beta = 12.3, 95% CI 3.84-20.8). It is concluded that subjects occupationally exposed to lead show a substantial compensatory increase in PTH and calcitriol activities which keep serum calcium levels within normal range. This may be of clinical significance since a sustained increase in calcitropic hormones in susceptible subjects may eventually increase the risk of bone disorders.     

A survey of the natural occurrence of aflatoxins and zearalenone in Argentine field maize: 1983-1994

In a Czech plant near Prague, 10 samples from male workers occupationally exposed to 1,3-butadiene and 13 exposed to 1,3-butadiene/styrene were compared with unexposed male negative controls, matched for age and smoking habits, for the presence of ras oncoproteins in their plasma. Proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence, using monoclonal ras antibody as the primary antibody. There were no statistically significant differences between the 3 groups (pooled two-sample t-test, untransformed and non-parametric Mann-Whitney test). These results are in keeping with the lack of exposure-related effects for 3 cytogenetic endpoints (chromosome aberrations, sister chromatid exchanges and micronuclei) already reported (Sorsa et al., 1994 Mutation Res., 309, 321-326) for this work-force exposed to low (below 3 ppm) exposure levels.     

Intraparticle mass transfer in high-speed chromatography of proteins

Styrene 7,8-oxide and ethylene oxide are widely used genotoxic bulk chemicals, which have been associated with potential carcinogenic hazard for occupationally exposed workers. Both epoxides alkylate DNA preferentially at the N-7 position of guanine and consequently produce single-strand breaks and alkali labile sites in the DNA of exposed cells. In order to study the role of human microsomal epoxide hydrolase (hmEH) in protecting cells against genotoxicity of styrene 7,8-oxide and ethylene oxide, we expressed the cDNA of hmEH in V79 Chinese hamster cells. We obtained a number of cell clones that expressed functionally active epoxide hydrolase. Among these, the clone 92hmEH-V79 revealed an especially high enzymatic mEH activity toward styrene 7,8-oxide (10 nmol converted per mg of protein per min, measured in the 9,000 x g supernatant of the cell homogenate), that was 100 times higher than that determined in mock-transfected cells and within the range of mEH activity in human liver. Styrene 7,8-oxide-induced DNA single-strand breaks/alkali labile sites (dose range 10 microM to 1 mM styrene 7,8-oxide) measured by the alkaline elution technique were significantly lower in the 92hmEH-V79 cells as compared to the mock-transfected cells. The protection against styrene 7,8-oxide genotoxicity in 92hmEH-V79 cells could be abolished by addition of valpromide, a selective inhibitor of microsomal epoxide hydrolase. These results clearly show that the metabolism of styrene 7,8-oxide by hmEH in 92hmEH-V79 cells was responsible for the protection against styrene 7,8-oxide genotoxicity. On the other hand, no protective effect of epoxide hydrolase expression could be observed on ethylene oxide-induced DNA damage with the recombinant cell line over a dose range of 0.5-2.5 mM ethylene oxide. This selectivity of the protective effect on epoxide genotoxicity thus appears to be an important factor that must be taken into account for the prediction of the genotoxic risk of epoxides themselves or compounds that can be metabolically activated to epoxides.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.     

Indications and dangers of osteosynthesis in childhood

The relationship between long-term occupational styrene exposure and disturbances in psychological functions was studied. The exposed group consisted of 98 male workers exposed to styrene in the manufacture of polyester plastic products, and the comparison group comprised 43 concrete reinforcement workers. The duration of exposure in years and the mean mandelic acid concentration in urine (five determinations during 5 weeks before the clinical examination) and the cumulative dose of exposure were used as the indicators of exposure. The psychological functions studied were intelligence, visuomotor speed, visuomotor accuracy, memory, vigilance, and psychomotor performance. Some personality test variables were also included. In a group comparison two variables (visuomotor inaccuracy and poor psychomotor performance) out of 20 showed statistically significant differences and characterized the styrene exposed group. These findings were confirmed when workers with low and high styrene exposure were compared. One variable measuring visuomotor speed and another measuring visual memory were related to the duration of exposure (R = 0.28, p less than 0.05). In a multiple regression analysis disturbances in visuomotor accuracy, poor psychomotor performance, and lowered vigilance proved to have some connection with a high mandelic acid concentration (R = 0.49, p less than 0.01). In addition workers with disturbances in visuomotor accuracy and slight disturbances in virumotor speed had higher mandelic acid concentrations when compared with those workers without any deterioration. The results indicate that disturbances in visuomotor accuracy and, to a lesser degree, in psychomotor performance are the main findings measured by test methods used in this study and related to one indicator of styrene exposure, mandelic acid concentration. The psychological methods used revealed subclinical symptoms related to exposure, and they should aid in making group diagnoses, e.g., when additional information is needed for the determination of the threshold limit value. The deterioration found in visuomotor accuracy could be related to lowered safety in work and traffic.     

Clinical, laboratory, phototest and phototherapy findings in polymorphic light eruptions: a retrospective study of 133 patients

OBJECTIVE: To study the blood benzene levels resulting from environmental and occupational benzene exposure. METHODS: Benzene in venous blood was measured in 243 nonoccupationally exposed subjects ("normal" people) and in 167 workers occupationally exposed to benzene. All exposed workers gave blood samples at the end of the work shift and on the following morning before resuming work. Blood benzene was assayed by gas chromatography (GC)-mass spectrometry. Occupational benzene exposure was monitored by environmental personal samplers and measured by GC analysis. RESULTS: The mean occupational benzene exposure for all 167 workers studied was 186 ng/l (58 ppb; range 5 1535 ng/l, 2-500 ppb). Overall, the mean blood benzene level of all workers was 420 ng/1 at the end of the shift and 287 ng/l on the morning thereafter. The blood benzene levels measured the morning after turned out to be significantly lower (t=3.6; P < 0.0001) than those measured at the end of the shift. The mean blood benzene level of the 243 "normal" subjects was 165 ng/l, which was significantly lower than that measured in the workers on the morning thereafter (t=5.8: P < 0.0000001). The mean blood benzene concentration was significantly higher in smokers than in nonsmokers in both the general population (264 versus 123 ng/l) and in the exposed workers. In the group of nonsmoking workers, whose workplace exposure to benzene was lower than 100 ng/l, blood benzene levels were similar (210-202 ng/l) to those measured in the nonsmoking general population (165 ng/l). End-of-shift blood benzene correlated significantly with environmental exposure (y=0.91x + 251; r=0.581; n=162; P < 0.00001). Finally, there was also a significant correlation between blood benzene measured at the end of the shift and that determined on the morning thereafter (y=0.45x + 109; r=0.572; n=156; P < 0.00001). Conclusion: Nonsmoking workers occupationally exposed to benzene at environmental levels lower than 100 ng/l (mean 35 ng/l) and the nonsmoking general population exposed to ubiquitous benzene pollution have similar blood benzene concentrations. This suggests that it is impossible to distinguish between occupational and environmental exposure when the benzene level in the workplace is less than 100 ng/l.     

Urinary mutagenicity as a biomarker in workers exposed to benzidine: correlation with urinary metabolites and urothelial DNA adducts

Urinary mutagenicity has been used in occupational and epidemiological studies for over two decades as a cost-effective, general biomarker of exposure to genotoxic agents. However, few studies have compared urinary mutagenicity to additional biomarkers determined among low- and high-exposed groups. To address this issue, we evaluated the relationship between urinary mutagenicity and other types of biomarkers in a cross-sectional study involving 15 workers exposed to the urinary bladder carcinogen benzidine (BZ, high exposure), 15 workers exposed to BZ-dyes (low exposure), and 13 unexposed controls in Ahmedabad, India. Urinary organics were extracted by C18/methanol and evaluated for mutagenicity in the presence of S9 in the Salmonella strain YG1024, which is a frameshift strain that overproduces acetyltransferase. The results were compared to biomarker data reported recently from the same urine samples (Rothman et al., Proc. Natl Acad. Sci. USA, 93, 5084-5089, 1996) that included a metabolite biomarker (the sum of the urinary levels of BZ + N-acetylbenzidine + N,N'-diacetylbenzidine) and a DNA adduct biomarker [a presumptive N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine (C8dG-ABZ) DNA adduct in exfoliated urothelial cells]. The mean +/- SE urinary mutagenicity (revertants/micromol of creatinine) of the low-exposure (BZ-dye) workers was 8.2 +/- 2.4, which was significantly different from the mean of the controls (2.8 +/- 0.7, P = 0.04) as was that of the mean of the high-exposure (BZ) workers (123.2 +/- 26.1, P < 0.0001). Urinary mutagenicity showed strong, positive correlations with urinary metabolites (r = 0.88, P < 0.0001) and the level of the presumptive C8dG-ABZ urothelial DNA adduct (r = 0.59, P = 0.0006). A strong association was found between tobacco use (bidi smoking) and urinary mutagenicity among the controls (r = 0.68, P = 0.01) but not among the exposed workers (r = 0.18, P = 0.11). This study confirms the ability of a biomarker such as urinary mutagenicity to detect low-dose exposures, identify additional genotoxic exposures among the controls, and correlate strongly with urinary metabolites and DNA adducts in the target tissue (urinary bladder epithelia) in humans.     

Photosensitized formation of 8-hydroxy-2'-deoxyguanosine and DNA strand breakage by a cationic meso-substituted porphyrin

Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA. In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic porphyrin. The data demonstrated a dose-dependent formation for each type of DNA damage. Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage. However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D2O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process. These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity.     

Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P-postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P-postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.