Methylmercury at low doses modulates the toxicity of PCB153 on PC12 neuronal cell line in asynchronous combination experiments.

Me-Hg and PCB153 are known neurotoxic contaminants which tend to accumulate in food, particularly in fish. Aim of this study was to perform asynchronous and combined exposure to Me-Hg and PCB153 in a neuronal rat cell line (PC12) to better characterise the antagonism observed at some combination concentrations. PC12 cells were treated with three concentrations of Me-Hg (0.1-0.5-1.0 microM) and PCB153 at one concentration (175 microM) in single and combined asynchronous exposures, using viability (MTT assay) as end-point. At all concentrations used, a statistically significant antagonistic effect was observed when Me-Hg preceded PCB153 exposure, while effect was additive when PCB153 preceded Me-Hg exposure. The antagonism is particularly evident at low concentrations of Me-Hg (0.1 microM). In conclusion, combined asynchronous exposure showed that whereas Me-Hg can modulate PCB153 toxicity, the opposite seems not to be true. Therefore, the use of asynchronous exposure could be a promising approach to study the mechanisms of toxicity of binary mixtures.

Antagonistic effects of methyl-mercury and PCB153 on PC12 cells after a combined and simultaneous exposure.

The study of interactions for those substances which tend to accumulate in food and affect the nervous system appears to be a fundamental point to characterize the combined exposure in vitro. In this study we included two food contaminants which are known neurotoxicants: methyl-mercury (Me-Hg) and the ortho-substituted PCB 153. PC12 cells were treated with Me-Hg (range 1e-7, 2e-6 M) and PCB153 (range 1e-5, 4e-4 M) in single and combined synchronous experiments and a mathematical model was set up according to the Loewe additivity criterion to evaluate the level of interaction between toxicants, using viability as end-point. At some concentrations (Me-Hg 5e-7 M and PCB153 1e-4 and 2e-4 M; Me-Hg 1e-6M and PCB153 5e-5 M; Me-Hg 1e-7 M and PCB153 4e-4 M), a statistically significant antagonist effect was observed. No interaction was observed for other combinations. The analysis of other toxicological parameters known to be modified in single exposure experiments (TBARS and intra-cellular dopamine) confirmed the viability results. The results of our work represent a starting point to generate novel information on the interactions between PCB153 and Me-Hg in vitro, as well as a new relevant experimental and mathematical approach useful to investigate the effects of different toxicant mixtures.

Analysis of oxidative stress in SK-N-MC neurons exposed to styrene-7,8-oxide.

Styrene-7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic volatile organic compound used industrially. Here we report the novel observation that several markers of oxidative stress were affected in SK-N-MC cells exposed for 16 h to concentrations of SO that induce apoptotic cell death. The production of Thiobarbituric Acid Reactive Substances (TBARS), rose from 69.1 +/- 15.7 nmol/g protein (control) to 119.3 +/- 39.2 and 102.0 +/- 17.3 nmol/g protein after exposure to 0.3 and 1 mM SO, respectively. Carbonyl group levels were significantly enhanced by SO at both concentrations. The lower dose also decreased sulphydryl groups. SO caused a marked oxidative DNA damage, as shown by a fivefold increase in 8-hydroxy-2(')-deoxyguanosine (8-OHdG). In addition, SO exposure resulted in alterations of scavenging abilities, given the reduction of both glutathione (GSH) and glutathione-S-transferase (GST) activity. Induction of expression of the oxidative stress response gene heme-oxygenase-1 (HO-1) and an increased HO-1 activity were also observed. These data provide compelling evidence that oxidative stress significantly contributes to SO toxicity in neuronal cells.

Styrene 7,8-oxide induces mitochondrial damage and oxidative stress in neurons.

Styrene 7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic compound used industrially. Neurons exposed to SO undergo apoptosis with characteristic features including chromatin rearrangements and caspase activation. We report that the execution phase of apoptosis induced by SO (0.3 mM) in SK-N-MC neurons is triggered by translocation of apoptogenic factors (e.g., cytochrome c) into the cytosol. In addition, mitochondria exhibit lower Ca2+ capacity and loss of mitochondrial membrane potential (DeltaPsi). Lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), is increased after 12 h. Pre-treatment with the antioxidant MnTBAP (100 microM) prevents the decrease of Ca2+ capacity, cytochrome c release, activation of caspases, exposure of phosphatidylserine and cell death. Hence, the neurotoxic effects of SO are related to mitochondrial damage and oxidative stress.

Dopamine (DA) metabolism in PC12 cells exposed to manganese (Mn) at different oxidation states.

The present study was aimed at assessing the role of Mn valency state in Mn-induced changes in DA metabolism by PC12 cells. Mn(ll)Cl2, Mn(lll)Acetate, and Mn(IV)O2 were used for these experiments. PC12 cells were incubated for 3, 24 and 72 hours to Mn nominal concentrations ranging from 10-8 to 10(-4) M in 24-well plates containing 2 x 10(5) cells/well. Supernatants and cellular materials were then separated and immediately processed for the analysis of dopamine (DA), and its metabolite 3,4-di-hydroxy-phenylacetic acid (DOPAC). Lactate dehydrogenase (LDH) activity and MTT cleavage were measured as indices of cell death. In parallel experiments, Mn-containing medium (10(-5) M) was removed and cells incubated for further periods with Mn-free medium to evaluate the reversibility of observed changes. At the end of the experimental periods, none of Mn-exposed cultures showed appreciable reduction in cell viability as compared to their respective controls. After exposure to Mn(II) and Mn(III), irreversible and dose-dependent decreases in the medium but not in intra-cellular DA were apparent. Indeed, 10(-4) M Mn(II) caused the disappearance of DA and DOPAC from the medium. The same effect was caused by 10(-5) M Mn(III), the dose-effect relationship being shifted towards lower dose levels. Mn(IV) induced a parallel and dose-dependent decrease of DA and DOPAC concentrations in both intra- and extra-cellular compartments. Such an effect was reversible after removal of Mn from the medium. Multiple interferences on DA metabolism are caused by Mn. Mn(II) and Mn(III) seem to block DA secretion without affecting DA turnover rate. Mn(IV) seems to cause DA depletion and aspecific (secondary) changes in secretion rates. Further studies are necessary to understand the mechanisms underlying the differential effects of various Mn compounds on DA metabolism.

Peripheral markers of neurochemical effects among styrene-exposed workers.

Monoamine oxidase B (MAO-B) activity in platelets, serum dopamine-beta-hydroxylase (DBH) activity, and serum prolactin (PRL) were measured during a cross-sectional investigation in workers occupationally exposed to styrene. The study group consisted of 53 workers (33 men and 20 women) employed for 9.3 years on average (range 1-22) in reinforced plastics plants. Sixty industrial workers with no known exposure to chemicals and comparable as to age, sex and confounding variables were recruited as controls. The activities of MAO-B in platelet-rich plasma and of DBH in serum from exposed and control subjects were measured within the same run, using methods based on the liquid-chromatographic determination of the reaction products. Serum PRL was determined by both EIA and RIA. Blood samples had been collected between 8:00 and 9:00 a.m. A lower DBH activity was found in exposed as compared to control workers (GM: 7.25 U/ml serum vs. 10.11 U/ml serum; p < 0.01), whereas MAO-B activity was significantly lower in a heavily exposed subgroup (10.1 vs. 13.8 U/10(7) platelets; p = 0.05), but not in the whole sample (p = 0.07). Serum PRL was higher both in male (GM: 8.90 ng/ml vs. 6.05 ng/ml; p < 0.01) and female (GM: 12.6 ng/ml vs. 9.33 ng/ml; p < 0.05) styrene-exposed workers as compared to their respective controls. Dose-response relationships were found for abnormally low DBH and abnormally high PRL values, with a threshold occurring at metabolite levels corresponding to 8h-TWA styrene concentrations in air around 25 ppm. In summary, this study shows that long-term exposure to relatively low levels of styrene can affect DBH activity and basal serum PRL. Owing to its sensitivity, PRL is a useful biomarker to show impairments of dopaminergic control on pituitary secretion. Since DBH is expression of catecholamine secretion, its decreased activity could represent an indirect index of altered turnover rate of the physiological substrate (i.e.dopamine) at the neuronal level. However, a direct interference by styrene metabolites on enzyme activity cannot be ruled out. Platelet MAO-B activity seems to be less sensitive to styrene exposure.

Styrene-induced changes in amacrine retinal cells: an experimental study in the rat.

Dopamine (DA) is synthesized in amacrine cells and released upon membrane depolarization in a calcium-dependent way. Thus, it is recognized to function as a major neurotransmitter or modulator in vertebrate retina. Owing to DA modulating activity on cone-horizontal cells transmission, depletion or dysfunction of amacrine cells could interfere with chromatic processing, accounting for the acquired dyschromatopsia described among styrene-exposed workers. The present study has been designed to test the hypothesis that amacrine cells represent a vulnerable target of styrene in subchronically exposed rats. Ten female Sprague-Dawley rats were exposed to 300 ppm styrene 6 h/day, 5 days/week, for 12 weeks; ten rats exposed to fresh air served as a control group. Whole mounted retinas were used for the morphometry of tyrosine hydroxylase (TH) immunoreactive cells (IR). DA content and TH activity were measured by HPLC and electrochemical detection and glutathione (GSH) was measured by HPLC tandem mass spectrometry (LC-MS/MS). In treated rats, morphometric analysis showed a loss of TH-IR amacrine cells (6.2/mm2 vs. 8.7/mm2 recorded in controls, p = 0.002), without any peripheral-central variation in cell loss. DA content was also lower in exposed, as compared to control animals (208.64 vs. 267.98 microg/g w.w., p = 0.004). The activity of TH in the whole retina was similar in styrene-exposed and control rats when expressed as a function of the wet weight, whereas it was much higher in styrene-exposed rats (+64%) when expressed as a function of the number of TH-IR amacrine cells (p < 0.001). Finally, retinal GSH was reduced by 30% in exposed as compared to control rats (p = 0.01). In summary, retinal TH-IR cells were sensitive to styrene exposure, which seems to cause both structural and functional changes, represented by cell loss and DA depletion, respectively. These findings confirm the vulnerability of dopaminergic systems to styrene toxicity, providing some insights on the possible mechanism of loss in chromatic discrimination recorded among workers occupationally-exposed to styrene.

Peripheral markers of catecholamine metabolism among workers occupationally exposed to manganese (Mn).

In a preliminary study of 11 men randomly selected in a ferro-alloy plant and of 15 control subjects, platelet monoamine oxidase (MAO) and serum dopamine beta-hydroxylase (DBH) activities were measured. A tendency towards lower MAO-B activity in the exposed workers as compared to control subjects (t = 1.95; P = 0.06) was found whereas DBH activity was similar. In the exposed group, a dose-effect relationship was noted between a manganese (Mn) cumulative exposure index (CEI) and DBH activity (R2 = 0.40, P < 0.05). Since DBH is an expression of catecholamine release, the relative increase in such activity could be envisaged as a compensatory mechanism to a reduced turnover rate as reflected by MAO-B activity. Owing to the limited sample size, these findings should be confirmed by further epidemiological and experimental studies.

Urban air pollution: use of different mutagenicity assays to evaluate environmental genetic hazard.

The genotoxic activities associated with airborne particulate matter collected in Parma (northern Italy) have been determined. The airborne particle extracts were tested for mutagenicity using Salmonella frameshift (TA98) and base-substitution (TA100) tester strains with and without S9 microsomal activation and Saccharomyces cerevisiae strain D7 in order to determine the frequency of mitotic gene conversion and ilv1-92 mutant reversion in cells harvested at stationary and logarithmic growth phase. The relationship between mitochondrial DNA mutations and ageing, degenerative diseases and cancer prompted us to take into account the mitochondrial informational target, i.e., the respiratory-deficient (RD) mutants. The results obtained show a variability in the response for the different test systems during different months. The Salmonella mutagenicity trend was directly correlated with carbon monoxide, nitrogen oxides (NOx) and Pb concentration in airborne particulates and inversely correlated with temperature, whereas the mitochondrial genotoxic effect was higher during spring and late summer. These data suggest that the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested.

An in vitro model for the assessment of manganese neurotoxicity.

PC12 (undifferentiated and differentiated) and C6 cells have been used to investigate kinetics, morphological and functional endpoints following exposure to MnCl(2) and manganic transferrin (Mn-Tf). [Mn](i) in undifferentiated (non-differentiated cells) exposed to both free (MnCl(2)) and bound Mn (Mn-Tf), was three- to fivefold lower as compared to differentiated (differentiated) PC12 cells and higher by one order of magnitude as compared to glial C6 cells. Exposure to both MnCl(2) and Mn-Tf was followed by time- and dose-dependent morphological changes characteristic of apoptosis, which was never observed in Mn-exposed C6 glial cells. Results from cell viability assays were consistent with apoptotic response rates quantified by cell count. Threshold concentrations for undifferentiated and differentiated PC12 cells were 10(-6) and 10(-5)m, respectively. Thus, despite their greater ability to accumulate Mn, differentiated PC12 cells are less sensitive to Mn-induced apoptosis. This model might be relevant to neuronal degeneration induced by Mn occurring in the developing brain and possibly in clinical manganism. Such critical doses at the cellular level seem to be consistent with Mn levels (5x10(-6)m) recorded in the basal ganglia of monkeys chronically exposed to Mn and developing clinical signs of manganism.

Serum prolactin in subjects occupationally exposed to manganese.

To evaluate whether or not occupational exposure to manganese (Mn) affects basal levels of serum prolactin (PRL), a cross-sectional study was carried out in 31 occupationally-exposed workers, aged 39.2 years (DS 7.9) exposed to manganese (Mn) dusts for 14.5 years (range: 5 to 29 years) in a ferroalloy producing plant. Thirty-four industrial workers not exposed to neurotoxic chemicals and of comparable age composed the control group. Airborne Mn concentrations in dusts of the furnace area ranged 210 to 980 micrograms/m3, which is below the current American Conference of Governmental Industrial Hygienists (ACGIH)-recommended threshold limit value-time weighted average (TLV-TWA) of 1 mg/m3. Manganese concentrations in blood Mn (MnB) and in urine (MnU) were significantly higher in Mn-exposed workers as compared to control workers. The Mn-exposed workers showed significantly higher serum prolactin (PRL) levels with the geometric mean (GM) being 9.77 ng/ml with a geometric standard deviation (GSD) of 1.69 as compared to controls (GM 4.65 ng/ml, GSD 1.78, p < 0.001). Serum PRL was negatively related to age and positively correlated with both MnB and MnU. Dose-effect relationships were still significant in partial correlation analysis after control for age. The prevalence of abnormally high PRL values was consistent with a dose-response relationship. The observed increase in serum PRL among Mn-exposed workers suggests an impairment of tonic inhibition by tubero-infundibular dopaminergic neurons. The correlation between PRL and both MnB and MnU in samples collected at least 48 h from the last exposure suggests that such indices provide an estimation of the target dose.

Development and validation of new screening tests for nephrotoxic effects.

Within the framework of an European Commission-funded project, groups of industrial workers exposed to heavy metals (cadmium, mercury and lead) or solvents were studied together with corresponding control groups. Eighty-one measurements were carried out on urine and serum samples and the scientific results together with individual questionnaire information were entered into a central database. Data obtained was assessed centrally and individually in subsidiary studies. The measurable contributions were assessed either singly or in combination, of smoking, gender, metal exposure and site, to nephrotoxicity. The potential value of each test as an indicator of nephrotoxicity was then assessed on the basis of sensitivity and specificity. A number of new tests including prostaglandins and for extracellular matrix components were investigated as well as established tests for renal damage and dysfunction. The data obtained from this comprehensive study emphasises the value of noninvasive biomarkers for the early detection of nephrotoxicity due to environmental toxins. The urinary profile varied with the type of environmental/occupational toxin. By careful selection of a small panel of markers they can be used to indicate the presence of renal damage, the principal region affected, and to monitor the progress of disease and damage. Biomarkers were also used to confirm and tentatively establish safe exposure levels to nephrotoxins.

[Definition of neurotoxic risk based on the benchmark dose in vitro: illustration with experimental models based on the use of known neurotoxic substances]. / Definizione del rischio neurotossico in base alla dose di riferimento ("benchmark dose") in vitro: esemplificazioni con modelli sperimentali basati sull'uso di sostanze neurotossiche note.

The BMD approach has been used to compare the cell viability (MTT assay) of different rat (C6 and PC12, glial and neuronal, respectively) and human cell lines (D384 and SK-N-MC, glial and neuronal, respectively) after 24-h exposure to the following neurotoxic substances: Manganese Chloride (MnCl2), Methyl-mercury (Me-Hg) and the enantiomers of Styrene Oxide (SO). For all rat and human cell lines, the potency of the examined compounds was: MnCl2 < S-SO < R-SO < Me-Hg. A preliminary comparison with in vivo toxicity data for these substances gave rise to consistent results. Whereas a reasonable agreement between in vitro and in vivo data has been found for Mn and styrene oxide, a wide scatter of LOAEL has been reported for Me-Hg and these appear to be either much higher or lower than the BMD for the MTT assay we observed in vitro.

Polymorphism of quinone-metabolizing enzymes and susceptibility to ozone-induced acute effects.

The role of the genetic polymorphism of NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferase micro-1 (GSTM1) in the responsiveness to O(3)-induced acute effects was investigated in 24 healthy nonsmokers performing 2-h bike rides at ambient O(3) varying from 32 to 103 ppb. Before and after rides, each subject performed spirometric tests and provided a blood sample for the measurement of the Clara cell protein CC16. NQO1 and GSTM1 polymorphisms were characterized by polymerase chain reaction- based methods. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct was also measured in DNA of peripheral leukocytes. Rides at O(3) > 80 ppb resulted in significant decrements of pulmonary function tests and increased levels of serum CC16, consistent with mild impairment in respiratory function and increased lung epithelial permeability, respectively. Whereas NQO1wt and GSTM1null subjects showed both functional changes and increased serum CC16 after acute O(3) exposure, people with other haplotypes showed a rise in serum CC16 but no changes in lung function tests. In NQO1wt and GSTM1null subjects, partial correlation analysis showed that functional decrements and increased serum CC16 are closely associated with each other and with O(3) levels, whereas no such relationships were found among subjects bearing other haplotypes. An increased reaction rate between O(3) and hydroquinones would be consistent with the greater increase in 8-OHdG after O(3) exposure in this "susceptible" group.

Peripheral markers of catecholaminergic dysfunction and symptoms of neurotoxicity among styrene-exposed workers.

AIM: A cross-sectional investigation was carried out to assess possible relations between styrene-induced changes in three peripheral markers of catecholaminergic dysfunction and self-reported symptoms of neurotoxicity. SUBJECTS: Male workers (n = 46) aged 14-60 (mean 29.5) years who had been exposed to styrene for an average of 6 (0.2-29) years were recruited in glassfiber reinforced plastics plants. A control group of 30 blue-collar workers aged 22-52 (mean 35) years and with no history of exposure to chemicals was recruited from local industries. Styrene exposure ranged from 5 to 120 ppm (8 h-TWA), the median level being relatively low (25 ppm, 8 h-TWA). Styrene metabolites, mandelic and phenylglycoxylic acids (MAPGA) in the "next morning" urine spot samples ranged from 32.0 to 931.1 mg/g creatinine (median 186.5). METHODS: Platelet monoamine oxidases B (MAO B) and dopamine beta-hydroxylase (DBH) activities were assessed using methods based on HPLC and electrochemical detection. Plasma prolactin (PRL) was measured by a commercially available immunoassay. Questionnaire 16 (Q16) was used to survey self-reported symptoms. RESULTS: Although there was no difference in DBH activity between exposed workers and controls, the most highly exposed workers had significantly lower activity than control subjects. A tendency to lower platelet MAO B activity in exposed than in control subjects was observed. The prevalence of plasma DBH and platelet MAO B values below the lower reference limit was similar in the two groups. PRL values exceeding the upper reference limit were higher (14/46 vs 2/30) among styrene-exposed workers, who also exhibited significantly higher median levels (10.0 vs 5.7 micrograms/l) than control subjects. Although the number of reported symptoms was similar among exposed and control subjects, in the exposed group it was positively associated with urinary MAPGA (Rho = 0.30, P = 0.04). Of the three peripheral markers of catecholaminergic dysfunction, plasma DBH was the only parameter negatively related to both urinary MAPGA (F = 9.56, P = 0.003) and the number of reported symptoms (Rho = 0.23, P = 0.05). CONCLUSIONS: Plasma PRL appears to be a sensitive marker of styrene-induced tubero-infundibular dopaminergic dysfunction in male subjects. DBH in plasma and MAO B in platelets seem to be less suitable markers for biomonitoring effect at the individual level, although DBH was related to the number of reported symptoms and to internal dose. Further studies on a larger and more exposed population are necessary to clarify the significance of these markers for health and their predictive value with regard to both subjective disturbances and concurrently administered performance tests.