Placental and lactational transfer of lead in rats: a study on the lactational process and effects on offspring

 The effects of placental and lactational exposure to lead (Pb) were studied in suckling rats after long-term exposure of their dams to Pb in drinking water. Dams were given 12 mM Pb-acetate in the drinking water 8 weeks prior to mating and during gestation. One group of dams was also continuously exposed during lactation until day 15. Neonates from Pb-treated dams were cross-fostered at birth to control dams treated with Na-acetate (12 mM) in the drinking water. In the same way, neonates from dams receiving control water were in the same way cross-fostered to Pb-exposed dams. All animals were killed at day 15 of lactation, when measurements were performed. Continuous Pb exposure during gestation and lactation resulted in milk Pb levels approximately 2.5 times higher than the blood Pb levels. When Pb exposure was terminated at parturition the milk Pb levels were at a level similar to those of blood Pb at day 15 of lactation, and only 10% of the milk levels found after continuous Pb exposure. Exposure to Pb via placenta and milk in offspring from dams exposed continuously resulted in more than 6 times higher blood and brain Pb levels than in offspring exposed only via the placenta. Exposure only via milk in offspring from dams exposed to Pb until parturition resulted in higher blood Pb levels than in offspring exposed to Pb only via the placenta. This indicates that the lactational transfer after current or recent exposure of Pb in dams is considerably higher than placental transfer. Offspring in all the exposed groups had decreased ALAD activity in the blood. An exponential relationship between blood Pb levels and ALAD activity was demonstrated in the offspring. Due to the exponential decrease in ALAD activity at increasing blood Pb levels, ALAD is particularly sensitive in reflecting differences in Pb exposure within the lowest range of blood Pb levels. There was a slight effect on weight gain in the offspring. However, there was no effect on milk quality, as measured by milk lipid, protein and calcium concentrations, nor on milk production assessed by the mammary gland RNA and DNA content. This indicates that the effect on weight gain was a direct effect of Pb in the offspring. Received: 4 January 1995/Accepted: 10 March 1995     

Evaluation of Bioadhesive Capacity and Immunoadjuvant Properties of Vitamin B<Subscript>12</Subscript>-Gantrez Nanoparticles

Purpose  To design bioadhesive Gantrez AN (poly[methyl vinyl ether-co-maleic anhydride], PVM/MA) nanoparticles (NP) coated with vitamin B₁₂ (Vit B₁₂), and investigate their application in oral antigen delivery. Methods  The association of Vit B₁₂ to Gantrez AN nanoparticles was performed by the direct attachment of reactive Vit B₁₂ to the surface of the nanoparticles (NPB), or linking to the copolymer chains in dimethylformamide prior to NP formation (NPB-DMF). Nanoparticles were characterized by measuring the size, zeta potential, Vit B₁₂ association efficacy, and stability of Vit B₁₂ on the surface of the nanoparticles. In vivo bioadhesion study was performed by the oral administration of fluorescently-labeled nanoparticle formulations to rats. Both systemic and mucosal immune responses were evaluated after oral and subcutaneous immunization with ovalbumin (OVA) containing Vit B₁₂-coated nanoparticles. Results  The Vit B₁₂ nanoparticles displayed homogenous size distribution with a mean diameter of about 200 nm and a negative surface charge. The association efficiency of Vit B₁₂ to NPB-DMF formulation was about two times higher than to the NPB, showing also a higher surface stability of Vit B₁₂. The bioadhesion study demonstrated that NPB-DMF had an important tropism to the distal portions of the gut, which was about two and 3.5 times higher than the tropism observed for NPB and control NP, respectively (p < 0.05). Oral administration of OVA-NPB-DMF induced also stronger and more balanced serum anti-OVA titers of IgG2a (Th1) and IgG1 (Th2) compared to control OVA-NP. In addition, oral immunization with OVA-NPB-DMF induced a higher mucosal IgA response than subcutaneous administration. Conclusions  These results indicate the benefits of bioadhesive Vit B₁₂-coated nanoparticles in oral antigen delivery eliciting systemic and mucosal immune response.     

In vivo and in vitro exposures for the evaluation of the genotoxic effects of lead on the Neotropical freshwater fish Prochilodus lineatus

In the present study, in vivo and in vitro exposures were used to assess the genotoxicity of lead (Pb) to the freshwater fish Prochilodus lineatus. The comet assay using blood, liver and gill cells, and the occurrence of micronuclei (MN) and other erythrocytic nuclear abnormalities (ENA) were used to assess the genotoxic potential of lead in vivo. Metallothionein content (MT) was measured in fish liver in order to evaluate the protection of fish against Pb toxicity. Fish erythrocytes were exposed to Pb in vitro (1, 3 and 6 h) and the number of viable cells, DNA integrity, using the comet assay, and lysosomal membrane stability, measured by the neutral red retention assay (NRRA) were analyzed. The results of the comet assay after in vivo toxicity tests (6, 24 and 96 h) showed that Pb was genotoxic for all the three tissues analyzed after 96 h exposure. A significant increase in liver MT content was observed after 6 and 24 h of Pb exposure. MN frequency did not increase after Pb exposures, but the frequency of the other ENA, such as kidney-shaped nuclei, segmented nuclei and lobed nuclei, showed a significant increase after 24 and 96 h, indicating that ENA is a better biomarker for Pb exposure than MN alone after short-term exposures. The results of the comet assay performed with erythrocytes in vitro exposed to lead confirmed its genotoxic effect and showed that DNA damage increased with increasing exposure time. Moreover, the NRRA clearly indicated that Pb induces a destabilization of the lysosomal membrane. These results demonstrate the potential genotoxicity and cytotoxicity of lead after acute exposures.     

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200?µM of H₂O₂ and evaluated the cytoprotective effects of Vit E (5?µM) and folate (0.01?µM) in H₂O₂-treated cells for 24?h. Following the exposure of endometrial cells to H₂O₂ alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H₂O₂ treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H₂O₂ toxicity. An increasing number of alive cells was showed in the cells exposed to H₂O₂ (50?µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.     

Comparative study on the induction of complex genomic alterations after exposure of mammalian cells to carboplatin and oxaliplatin

Metal complexes are still broadly used as the first line of the treatment for different types of tumors nowadays. Carboplatin and oxaliplatin were authorized for clinical use, even though there is little information on the mutagenic profile associated to their usage. This study evaluated the cytostatic effects and the induction of complex genomic alterations after 24-h treatment of CHO-K1 cells to concentrations of 12.5–800?μM of carboplatin and oxaliplatin in the cytokinesis-block micronucleus assay (CBMN-Cyt). The results demonstrated that carboplatin and oxaliplatin significantly increased the frequency of micronuclei (MN), nucleoplasmatic bridges (NPBs), and nuclear buds (NBUDs). On one hand, oxaliplatin induces significantly more chromosomal abnormalities than carboplatin at concentrations of 12.5 and 25?μM. On the other hand, carboplatin, in cells exposed to concentrations of 50 and 100?μM, is more efficient than oxaliplatin in the induction of chromosomal instability events. Both drugs cause significant reduction in the cytokinesis-block proliferation index, demonstrating their cytostatic effects at concentrations 50–800?μM. The results of this study shed more light on the characterization of biological effects associated with the exposure to carboplatin and oxaliplatin.     

Effects of lead on lactating rats and their sucklings

Lead was administered to lactating rats in drinking water (0, 1, 10 and 100 ppm) from the day of delivery up to day 21 after delivery, at which time the mothers and their newborns were sacrificed. Various parameters of blood: lead concentration (PbB), hematocrit (Htc), hemoglobin (Hb), free erythrocyte porphyrin concentration (FEP), δ-aminolevulinate dehydratase activity (ALAD) — and of tissue: ALAD activity, free tissue porphyrin concentration (FTP) and lead concentration (PbT) were determined.In mothers, a significant increase of PbB and a reduction in ALAD activity of blood were found in the 100 ppm group. In tissues Pb was significantly increased in liver of the 100 ppm group and in kidney of the 10 and 100 ppm groups. None of the biochemical parameters measured in tissues was significantly modified.In suckling rats an increase in PbB and a reduction of ALAD activity in blood were found in the 10 and 100 ppm lead groups. Pb concentration was significantly increased in liver, kidney and brain of the 100 ppm group and in kidney of the 10 ppm group. Lead storage in kidney of the 100 ppm group was associated with a marked increase in FTP and a slight reduction in ALAD activity.On the basis of the biochemical parameters studied in the newborn rats, the “no-effect” level of lead administered in drinking water during lactation is around 1 ppm, which is rather similar to that found when lead was administered to the mother before and/or during pregnancy.     

Assessment of the genotoxicity and cytotoxicity in environmentally exposed human populations to heavy metals using the cytokinesis‐block micronucleus cytome assay

The cytokinesis‐block micronucleus cytome (CBMN‐Cyt) assay was developed as a system for evaluating DNA damage, cytostasis, and cytotoxicity. The aim of the present study was to estimate levels of micronuclei (MNi), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (apoptosis/necrosis), nuclear division index, and nuclear division cytotoxicity index values in the peripheral blood lymphocytes of environmentally exposed subjects to heavy metals from five Bosnian regions, characterized by different exposure to heavy metals. The study was performed using CBMN‐Cyt assay, considering factors, such as age, gender and smoking habits and their possible effects on analyzed parameters. In total, 104 healthy subjects were selected (49.04% females and 50.96% males; average age, 35.41 years; 51.92% smokers and 48.08% nonsmokers). There was significant difference between the frequency of NBUDs in Tuzla as compared to the control group. Furthermore, there was observed a statistically significant difference for the frequency of NPBs between Zenica, Olovo, and Kakanj when compared with the controls. Males showed a significantly higher number of apoptotic cells than females in controls. There were significant differences between smokers and nonsmokers in the frequency of NPBs in controls (higher in nonsmokers) and necrotic cells in Olovo (higher in nonsmokers). The pack years of smoking significantly influenced the number of necrotic cells in controls and the frequency of NBUDs in the overall sample. The results of the present study provide evidence of significantly increased frequency of NPBs and NBUDs in exposed subjects, suggesting that these endpoints are highly sensitive markers for measuring genotoxicity. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1331–1342, 2015.     

The Relationship between Blood and Serum Lead Levels in Peripartum Women and their Respective Umbilical Cords

Abstract: Foetal exposure to lead (Pb) during pregnancy is a major problem. However, no previous study has examined whether Pb concentrations in blood (Pb‐B) and in serum (Pb‐S) from pregnant women correlate with Pb‐B and Pb‐S in the foetuses. This hypothesis was tested in the present study. We measured Pb‐B and Pb‐S in 120 healthy pregnant women (more than 38 weeks of gestation) and their respective umbilical cord samples. The analyses were carried out with an inductively coupled plasma mass spectrometer. We found higher Pb‐B levels in the women compared with their respective umbilical cord samples (1.736 ± 0.090 μg/dL and 1.194 ± 0.062 μg/dL, respectively; p < 0.05). In parallel, we found higher Pb‐S levels in the women compared with their respective umbilical cord samples (0.042 ± 0.003 μg/dL and 0.032 ± 0.003 μg/dL, respectively; p < 0.05). However, similar %Pb‐S/Pb‐B ratios were found in the women compared with their respective umbilical cord samples (2.414 ± 0.210% and 2.740 ± 0.219%, respectively; p > 0.05). Interestingly, we found positive correlations between Pb‐B in the umbilical cords and Pb‐B in the respective pregnant women (rs = 0.5714; p < 0.0001), and between Pb‐S in the umbilical cords and Pb‐S in the respective pregnant women (rs = 0.3902; p < 0.0001) as well as between %Pb‐B/Pb‐S in the umbilical cords and %Pb‐B/Pb‐S in the respective pregnant women (rs = 0.3767; p < 0.0001). These results indicate that the assessment of Pb‐B and Pb‐S in pregnant women provides relevant indexes of foetal exposure to Pb. Moreover, the similar %Pb‐S/Pb‐B in pregnant women and in the umbilical cords shows that the foetuses are directly exposed to the rapidly exchangeable Pb fraction found in their mothers.     

Strategies for Hepatic Gene Correction

Many different cancer types have previously been found to show increased uptake of the vitamins folate, vitamin B12, and biotin; however, it is not known whether these tumor lines show increased uptake of one or more of the vitamins. The current study was designed to examine the relative uptake of the three vitamins in 10 different types of cell lines. Rhodamine-labeled hydroxypropyl-methacrylamide (HPMA) was targeted with vitamin B₁₂, folate, or biotin, and the uptake of the labeled polymer was compared both in in vitro cell cultures and in mice-bearing tumors from a variety of tumor cell lines. Fluorescent microscopy of cell cultures and histological examination of tumor sections showed greatly increased uptake of the fluorescently labeled polymer in many tumors when the polymer was targeted with folate, biotin, or vitamin B₁₂. Tumors with enhanced uptake of vitamin B₁₂- or folate-targeted rhodamine-HPMA also showed increased uptake of biotin-Rho-HPMA. In contrast, tumors with increased uptake of folate-Rho-HPMA did not show increased uptake of vitamin B12 (VB₁₂)-HPMA and vice versa. These findings suggest that vitamin-targeted polymers may greatly increase the uptake of drug–polymer complexes in certain tumors, which may result in an increased efficacy of antitumor agents, and which may allow for easier imaging of both the primary and metastatic tumors.     

Assessment of how pregnancy modifies plasma lead and plasma/whole blood lead ratio in ALAD 1-1 genotype women

Pregnant women are one of the most sensitive populations to the toxic effects associated with lead (Pb) exposure. These effects are primarily associated with plasma Pb (Pb-P), which reflects the most rapidly exchangeable fraction of Pb in the bloodstream, and elevated maternal Pb-P may be more relevant to foetal Pb exposure than whole blood Pb (Pb-B). We investigated how pregnancy affects Pb-B, Pb-P and %Pb-P/Pb-B ratios without the influence of the delta-aminolevulinic acid dehydratase (ALAD) G177C polymorphism, which is a major genetic factor influencing Pb-B, Pb-P and %Pb-P/Pb-B ratios. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion in nine pregnant and 20 non-pregnant women, aged 18-33, environmentally exposed to Pb. Here, we included only women with ALAD 1-1 genotype. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. We found no differences in Pb-B (P > 0.05). However, pregnant women had a 2-fold increase in Pb-P and a 3-fold increase in %Pb-P/Pb-B (both P < 0.01) compared to non-pregnant women. These alterations in Pb concentrations associated with pregnancy are similar to those associated with different ALAD gene variants. We can now better appreciate how pregnancy affects foetal exposure to Pb without the influence of this important genetic factor.     

Biological monitoring of workers exposed to lead stearate

This study was conducted to examine the usefulness of some of the commonly used biological parameters for monitoring of workers exposed to lead stearate. Forty-two lead stearate workers from a lead stabilizer factory and 26 workers exposed to inorganic lead compounds were involved in this study. Although the workers had similar blood lead values (PbB), subjects exposed to lead stearate were found to have a significantly higher concentration of lead in plasma (PbP), 1.0 +/- 0.57 micrograms dl-1) than workers exposed to inorganic lead compounds (0.42 +/- 0.3). The ratio of PbP to PbB was ca. 2.5 times higher for lead stearate workers (0.38) than the inorganic lead workers (0.15). These data suggest that the different chemical properties of lead stearate may result in different distributional patterns of the metal in different blood components. On the other hand, the activity of delta-aminolevulinic acid dehydratase (ALAD), an enzyme highly sensitive to lead, was not so much depressed among the lead stearate workers as that of workers handling inorganic lead. A poor correlation was also observed between PbB and ALAD activity of the stearate workers. These findings indicate that PbB and ALAD are not good biological indicators for evaluating the toxicological effect of lead stearate exposure.     

Evidence for specific lead-delta-aminolevulinate complex formation by carbon-13 nuclear magnetic resonance spectroscopy.

Lead is known to have toxic effects on heme synthesis, being an especially potent, and, relative to other metals, specific inhibitor of the enzyme δ-aminolevulinate dehydratase (ALAD). The mechanism of inhibition of ALAD by lead is uncertain, but binding of lead to ALAD sulfhydryl groups has been assumed. Mechanisms involving lead interaction with the enzyme substrate (ALA) rather than the enzyme are also reasonable, but have not as yet been considered; therefore the interaction of lead acetate with ALA was investigated by carbon-13 NMR spectroscopy, a technique shown to be very powerful in examining ligand-metal interactions. Addition of lead acetate solution to 0.25 m ALA at pH 6.5 resulted in a pronounced shift of certain of the carbon resonances of the latter, a maximal effect occurring at an ALA: Pb concentration ratio of 2. No similar effects were observed with acetates of calcium, zinc, cadmium, silver, or mercury. Studies on the variation in shift of resonances from invididual carbon atoms with lead acetate concentration suggested that lead interacted most strongly in the region of the carboxyl and α-carbon atoms of ALA. The variation of shift with pH showed that at an ALA: Pb ratio of 1 the pK_a of the ALA carboxyl group was decreased by 1.2 units. These studies demonstrate that lead is specific among the metals tested in forming a complex with δ-aminolevulinic acid. Such complex formation may be important in the mechanism of lead toxicity on the heme biosynthetic pathway.     

EP4 receptor signalling in immature B cells involves cAMP and NF-κB dependent pathways

Objectives Delineation of EP4 receptor signalling properties in immature B cells. Methods WEHI 231 cells were used as a model of immature B lymphocytes. The effects of PGE2, EP4 receptor antagonist, EP4 receptor agonist, forskolin and adenylate cyclase inhibitor on proliferation of WEHI 231 cells were examined by MTS assay. Cyclic adenosine monophosphate (cAMP) levels were examined by ELISA, whereas phosphorylation of vasodilator‐stimulated phosphoprotein (VASP), kinase, extracellular signal‐regulated kinase1/2, IκB‐α and nuclear factor (NF)‐κB subunit p105 were subjected to Western blot analysis. Translocation of NF‐κB subunit p65 and EPRAP (EP4 receptor associated protein) was examined by fluorescence microscopy. Levels of early growth response factor (Egr)‐1 mRNA were determined by quantitative PCR. Key findings We identified the EP4 receptor as the principal molecule mediating the growth‐suppressive effect of prostaglandin E2 in WEHI 231 cells. EP4 receptor activation results in cAMP formation and the activation of protein kinase A, NF‐κB1 p105 subunit stabilization and inhibition of IκBα phosphorylation, followed by the accumulation of NF‐κB p65 subunit in the cell cytoplasm, whereas the activation of PI3K is not involved in EP4 receptor signalling. Elevation of cAMP and inhibition of NF‐κB activation are two possible mechanisms by which the EP4 receptor inhibits the proliferation of immature B lymphocytes. Conclusions Modulation of the EP4 receptor on immature B lymphocytes provides important insight into the observed action of PGE2 and opens new possibilities for the development of therapies for autoimmune diseases, leukaemia and lymphomas.     

Cell-cycle analysis and micronuclei frequency reveals G0/G1 blockers as weak micronuclei inducers

Micronuclei (MN) formation is generally attributed to error in DNA synthesis or mitosis, which are represented by the S or G₂/M phase respectively, in the cell-cycle histogram. Interestingly, many of the known anticancer drugs target these cell-cycle phases to elicit cytotoxicity. Here, we attempted to identify whether any correlation exists between the cell-cycle effect and MN induction potential using various treatments. In addition, we tracked down MN in cycling cells to assess its final fate. We treated SiHa cells with various known drugs and correlated their effects on cell-cycle and MN frequency. MN-tracking studies were performed in peripheral mononuclear and siHa cells upon staining with Giemsa and ethidium bromide respectively. We observed MN induction by all the tested drugs irrespective of their basic effect on cell cycle. However, MN induction was more with drugs which interfere with the S or G₂/M than the G₀/G₁ phase. Our results indicate G₀/G₁ blockers to be comparatively safer drugs. Additionally, our results show that expulsion out of cells may be one of the main fates of drug-induced MN.     

The size‐dependent genotoxic potentials of titanium dioxide nanoparticles to endothelial cells

Despite intensive research activities, there are still many major knowledge gaps over the potential adverse effects of titanium dioxide nanoparticles (TiO₂‐NPs), one of the most widely produced and used nanoparticles, on human cardiovascular health and the underlying mechanisms. In the present study, alkaline comet assay and cytokinesis‐block micronucleus test were employed to determine the genotoxic potentials of four sizes (100, 50, 30, and 10 nm) of anatase TiO₂‐NPs to human umbilical vein endothelial cells (HUVECs) in culture. Also, the intracellular redox statuses were explored through the measurement of the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) with kits, respectively. Meanwhile, the protein levels of nuclear factor erythroid 2‐related factor 2 (Nrf2) were also detected by western blot. The results showed that at the exposed levels (1, 5, and 25 μg/mL), all the four sizes of TiO₂‐NPs could elicit an increase of both DNA damage and MN frequency in HUVECs in culture, with a positive dose‐dependent and negative size‐dependent effect relationship (T100 < T50 < T30 < T10). Also, increased levels of intracellular ROS, but decreased levels of GSH, were found in all the TiO₂‐NP‐treated groups. Intriguingly, a very similar manner of dose‐dependent and size‐dependent effect relationship was observed between the ROS test and both comet assay and MN test, but contrary to that of GSH assay. Correspondingly, the levels of Nrf2 protein were also elevated in the TiO₂‐NP‐exposed HUVECs, with an inversely size‐dependent effect relationship. These findings indicated that induction of oxidative stress and subsequent genotoxicity might be an important biological mechanism by which TiO₂‐NP exposure would cause detrimental effects to human cardiovascular health.     

Induction of micronuclei on Greek hairdressers occupationally exposed to chemical mixtures

Since the early 20th century, hairdressers (HD) have been exposed to a wide range of harmful chemical products. To determine the possible genetic damage to HD, as a result of their occupational exposure to combinations of different chemical factors, we applied the micronucleus assay on their peripheral blood lymphocytes cultures. The micronucleus assay was performed on blood samples from 20 Greek female HD and 20 control women, having no connection with the occupation, from the same area. In the results analysis, parameters included were age, smoking habits, and duration of occupational exposure. The results of our study showed a significant increase in HD micronuclei (MN) frequency, compared to the controls (13.4 ± 1.00 vs. 8.05 ± 0.65). The frequency of large‐size MN was significantly higher in the HD and presented potential correlation with the phenomenon of aneuploidy. A statistically significant difference in the frequency of MN between HD and controls who smoked was observed, while this was not the case with the non smoker groups. However, multiple regression analysis showed no significant correlation between smoking habits and MN frequency. The observed increase of the frequency of MN in HD is attributed to the long‐term occupational exposure of HD in combination with different chemical factors. Since in the literature there are very few similar studies, further combined studies are suggested on a larger number of HD from different countries, combining biological and molecular techniques, as well as chemical analytical methods of determining and tracing the chemical factors in both the occupational environment and their organisms. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Different pharmacological properties of the optical isomers of MN9202, a novel 1,4‐dihydropyridine Ca2+ channel modulator,in rat ventricular myocytes

1. We have shown previously that 1,4‐dihydro‐2,6‐dimethyl‐4‐(3‐nitrophenyl)‐3,5‐pyridinedicarboxylic acid pentyl methyl ester (MN9202), a new 1,4‐dihydropyridine Ca²⁺ channel modulator, has significant hypotensive effects and favourable pharmacokinetic characteristics. As a chiral molecule, MN9202 has two optical isomers. The aim of the present study was to evaluate the pharmacological properties of the two enantiomers. 2. The two enantiomers, S‐(?)‐ and R‐(+)‐MN9202, were obtained by HPLC. At 1 μmol/L, both racemic MN9202 and S‐(?)‐MN9202 decreased the contractility of rat ventricular myocytes by 54.0 and 64.4%, respectively, compared with control, whereas R‐(+)‐MN9202 enhanced cell shortening by 10.1%. At 1 μmol/L, racemic MN9202 markedly reduced calcium transient (CaT) and L‐type Ca²⁺ channel current (I_Ca,L) by 60.0 and 50.7%, respectively, whereas the reductions in CaT and I_Ca,L produced by 1 μmol/L S‐(?)‐MN9202 were greater still (62.2 and 65.7%, respectively). In contrast, 1 μmol/L R‐(+)‐MN9202 increased CaT and I_Ca,L by 11.4 and 10.6%, respectively. Furthermore, findings from kinetics studies of I_Ca,L revealed that the steady state inactivation curve of I_Ca,L was shifted towards a hyperpolarizing potential by S‐(?)‐MN9202, but towards a depolarizing potential by R‐(+)‐MN9202. These results demonstrate different effects of R‐(+)‐MN9202 and S‐(?)‐MN9202. 3. In conclusion, the findings of the present study suggest that the chirality of MN9202 results in opposing pharmacological properties of its two enantiomers: S‐(?)‐MN9202 may be responsible for the therapeutic effects of racemic MN9202, whereas R‐(+)‐MN9202 contributes to it unwanted effects. The findings of the present study also indicate that MN9202 may be used as a new probe with which to investigate the structure–function relationships of Ca²⁺ channels.     

Serum Metabolomic Profiling of Sulphur Mustard‐Exposed Individuals Using 1HNuclear Magnetic Resonance Spectroscopy

Sulphur mustard is an alkylating agent that reacts with different cellular components, causing acute and delayed complications that may remain for decades after exposure. This study aimed to identify differentially expressed metabolites between mustard‐exposed individuals suffering from chronic complications compared with unexposed individuals as the control group. Serum samples were obtained from 15 mustard‐exposed individuals and 15 apparently healthy unexposed individuals. Metabolomic profiling was performed using ¹H nuclear magnetic resonance spectroscopy, and analyses were carried out using Chenomex and MATLAB softwares. Metabolites were identified using Human Metabolome Database, and the main metabolic pathways were identified using MetaboAnalyst software. Chemometric analysis of serum samples identified 11 differentially expressed metabolites between mustard‐exposed and unexposed groups. The main pathways that were influenced by sulphur mustard exposure were related to vitamin B₆ (down‐regulation), bile acid (up‐regulation) and tryptophan (down‐regulation) metabolism. Metabolism of vitamin B₆, bile acids and tryptophan are the most severely impaired pathways in individuals suffering from chronic mustard‐induced complications. These findings may find implications in the monitoring of exposed patients and identification of new therapeutic approaches.     

Evaluation of genotoxic effects in a group of workers exposed to low levels of styrene

Occupational exposure to styrene was studied in a group of workers engaged in the production of fiberglass-reinforced plastics. Sister-chromatid exchanges (SCE), micronuclei (MN), and DNA damage (evaluated by means of comet assay) were measured in peripheral blood cells from the exposed workers and from a control population. Mandelic acid concentration, an indicator of styrene exposure level, was measured in urine samples collected at the end of the work shift. Average estimated values for styrene exposure were slightly below the threshold limit value (TLV) of 20 ppm recommended by the American Conference of Governmental Industrial Hygienists. Significant increases (P< or =0.01) have been found for SCE and MN frequencies and comet tail length among exposed individuals, as well as significant decreases (P< or =0.01) in the proliferation indices, as compared with control population. High correlation has been obtained between endpoints evaluated and exposure length, and increased values of SCE and MN frequencies and comet tail length have been found among smokers only in the exposed population. The high correlation obtained among SCE and MN frequencies and comet tail length, and the increase of these parameters in the exposed group with regard to control group justify the use of these three biomarkers in the evaluation of genotoxic effects in human populations exposed to styrene.