Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans.

Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.

Characterization of the modes of action and dose-response relationship for thiocyanate on the thyroid hormone levels in rats using a computational approach.

Current practices for evaluating the cumulative risk of thyroid-active chemical mixtures (perchlorate, thiocyanate, nitrate) focus on the inhibition of thyroidal iodide uptake via the sodium iodide symporter (NIS) as the mode of action for potency equivalence calculations. However, unlike perchlorate, thiocyanate presents additional modes of action within the thyroid that could contribute to the overall thyroid perturbation. We tested the hypothesis of whether assuming a single mode of action of thyroidal iodide uptake inhibition is sufficient for describing the observed dose-response relationship for thiocyanate and its effects on serum thyroxine levels. An interaction model was developed by linking a biologically based dose-response model for iodide and thyroid hormones to a thiocyanate physiologically based pharmacokinetic model. Each model, adapted from the literature, was restructured and recalibrated in a Bayesian framework for the current mode of actions study. For a chronic exposure scenario, NIS inhibition alone was found not to be sufficient to describe the dose-response relationship for thiocyanate. Inclusion of additional modes of action involving iodide flux across the thyroid membrane and inhibition of iodide organification via thyroid peroxidase showed only moderate improvements in capturing the dose-response at environmental thiocyanate doses of exposure and failed to capture trends at very high doses. Our findings emphasize the need for more mechanistic data for chronic exposure scenarios to characterize better the overall dose-response relationship for thiocyanate. Risk assessment approaches for thyroid-active chemical mixtures that rely on NIS inhibition as the single mode of action may over-predict the contribution of thiocyanate to thyroid disruption.

Thiocyanate: a review and evaluation of the kinetics and the modes of action for thyroid hormone perturbations.

Exposure of the population to thiocyanate is predominantly through the diet and cigarette smoke. Thiocyanate is a potential thyroid disruptor due to its capacity to inhibit the uptake of iodide by the thyroid. Thiocyanate also interacts with the enzymatic reactions associated with iodide organification and thyroid hormone synthesis. Quantification of the dose-response relationships of thiocyanate and alteration in thyroid hormone levels is important for evaluating the risk of exposure to thiocyanate in humans. In this review, we highlight the key whole-body and intra-thyroidal aspects of thiocyanate kinetics in rats and its various modes of action for perturbing thyroid function. The inter-play between the various transporter- and enzyme-mediated modes of action contributes to the complexity in the dose-response relationship determinations for thiocyanate. We map the available modes of action in a mechanistic and quantitative manner. Findings summarized in this study can help support the development of a quantitative model to study the interaction effects of thiocyanate on the thyroid function. Additionally, the data gaps identified can help guide future experimental designs to characterize further thiocyanate dose-response. Finally, the strengths and weaknesses in current risk assessment considerations used for thiocyanate as a component of thyroid-active chemical mixtures are discussed.

The effect of smoking on placental pendrin expression.

Pendrin is important for transport of iodine across the placenta. Thiocyanate coming from cigarette is a competitive inhibitor of iodine transport. We aimed to evaluate the pendrin immunostaining intensity in placentas of smoker and non-smoker women. Placental tissues from 61 women, of which 28 were in smoking, and 33 were in non-smoking group were evaluated by immunohistochemical staining. Positive immunostaining was evaluated using a semiquantitative score: 0, negative; +, mild; ++, moderate; and +++, intense. Birth weight was significantly lower in the smoker group (p = 0.024). There was a negative correlation between birth weight and intensity of placental pendrin immunostaining in the smoker group (r = -0.44, p = 0.02). Placentas of the smoking women showed significantly higher immunostaining with pendrin than the control group (p = 0.006). Thiocyonate coming from cigarettes may competitively inhibit pendrin mediated iodine transport in the placenta and adversely affect foetal development by this mechanism.

CO-occurring exposure to perchlorate, nitrate and thiocyanate alters thyroid function in healthy pregnant women.

BACKGROUND: Adequate maternal thyroid function during pregnancy is necessary for normal fetal brain development, making pregnancy a critical window of vulnerability to thyroid disrupting insults. Sodium/iodide symporter (NIS) inhibitors, namely perchlorate, nitrate, and thiocyanate, have been shown individually to competitively inhibit uptake of iodine by the thyroid. Several epidemiologic studies examined the association between these individual exposures and thyroid function. Few studies have examined the effect of this chemical mixture on thyroid function during pregnancy OBJECTIVES: We examined the cross sectional association between urinary perchlorate, thiocyanate and nitrate concentrations and thyroid function among healthy pregnant women living in New York City using weighted quantile sum (WQS) regression. METHODS: We measured thyroid stimulating hormone (TSH) and free thyroxine (FreeT4) in blood samples; perchlorate, thiocyanate, nitrate and iodide in urine samples collected from 284 pregnant women at 12 (±2.8) weeks gestation. We examined associations between urinary analyte concentrations and TSH or FreeT4 using linear regression or WQS adjusting for gestational age, urinary iodide and creatinine. RESULTS: Individual analyte concentrations in urine were significantly correlated (Spearman's r 0.4-0.5, p<0.001). Linear regression analyses did not suggest associations between individual concentrations and thyroid function. The WQS revealed a significant positive association between the weighted sum of urinary concentrations of the three analytes and increased TSH. Perchlorate had the largest weight in the index, indicating the largest contribution to the WQS. CONCLUSIONS: Co-exposure to perchlorate, nitrate and thiocyanate may alter maternal thyroid function, specifically TSH, during pregnancy.

The smoking-associated oxidant hypothiocyanous acid induces endothelial nitric oxide synthase dysfunction.

Smokers have an elevated risk of cardiovascular disease but the origin(s) of this increased risk are incompletely defined. Considerable evidence supports an accumulation of the oxidant-generating enzyme MPO (myeloperoxidase) in the inflamed artery wall, and smokers have high levels of SCN(-), a preferred MPO substrate, with this resulting in HOSCN (hypothiocyanous acid) formation. We hypothesized that this thiol-specific oxidant may target the Zn(2+)-thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signalling molecule NO•. Decreased NO• bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smoking-associated disease. In the present study it is shown that exposure of isolated eNOS to HOSCN or MPO/H2O2/SCN(-) decreased active dimeric eNOS levels, and increased inactive monomer and Zn(2+) release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl(-)-treated samples. eNOS activity was increasingly compromised by MPO/H2O2/Cl(-) with increasing SCN(-) concentrations. Exposure of HCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl(-) with increasing SCN(-), increased eNOS monomerization and Zn(2+) release, and decreased activity. Intact HCAECs exposed to HOCl and HOSCN had decreased eNOS activity and NO2(-)/NO3(-) formation (products of NO• decomposition), and increased free Zn(2+). Exposure of isolated rat aortic rings to HOSCN resulted in thiol loss, and decreased eNOS activity and cGMP levels. Overall these data indicate that high SCN(-) levels, as seen in smokers, can increase HOSCN formation and enhance eNOS dysfunction in human endothelial cells, with this potentially contributing to increased atherogenesis in smokers.

Effect of maternal nicotine/thiocyanate exposure during gestational period upon pituitary, thyroid and parathyroid function/morphology of 1-month-old rat offspring.

INTRODUCTION: Impact of in utero exposure to nicotine, on the structure of the thyroid-pituitary axis and the parathyroid glands have been examined in 1-month-old rats and compared with that of thiocyanate. MATERIALS AND METHODS: Three pregnant female groups were used; control, nicotine and thiocyanate. Treatment started from gestation day (4-20) and the specimens were harvested from the male offspring of all groups at the age of 1 month and processed for light, electronmicroscopic and immunohistochemical examination. Total triiodothyronine (tT3), total thyroxine (tT4) and total thyrotropin (TSH) were quantitatively determined in serum. RESULTS: Both nicotine and thiocyanate activated the thyroid follicular cells, with an increase in height (about 30 %) and a negative feedback on the pituitary thyrotrophs which revealed a reduction in the number of cytoplasmic secretory granules, particularly the thiocyanate group. However, in thiocyanate group there was signs of impaired secretory activity of the thyroid gland. The arbitrary area of parathyroid chief cells, increased (about 45 %) particularly in nicotine group, with signs of reduced activity and a positive feedback on the parafollicular cells which revealed hypertrophy, proliferation (25 %) and increased intensity of positive immunohistochemical reaction for calcitonin. CONCLUSION: Nicotine impaired chief parathyroid cells activity and consequently activated parafollicular cells. Thiocyanate reduced pituitary thyrotrophs activity, whereas both nicotine and thiocyanate increased thyroid follicular cells activity. This impact of in utero exposure persisted for 1-month postnatal.

Toxicokinetic aspects of thiocyanate after oral exposure to cyanide in female Wistar rats in different physiological states.

Cyanide (CN) is an ion that has been well studied in toxicology and has been associated with several intoxication episodes: the ingestion of contaminated foods and water, chemical war, suicides, homicides, occupational exposures and the use of certain medicines. The aim of the present study was to determine the toxicokinetic parameters of thiocyante (SCN), the main metabolite of CN, after oral administration of potassium cyanide (KCN) to female rats at diestrus, gestational and lactational periods. Female Wistar rats were divided into three equal groups: virgins in the diestrus phase of the estrus cycle, females at the 14th day of gestation and females at the 14th day of lactation. Each group of rats received 3.0 mg of potassium cyanide per kilogram (KCN/kg body weight) by gavage, and blood was collected at several time points. We also collected amniotic fluid from pregnant rats and milk from the nursing rats to analyze thiocyanate concentration. The results showed that SCN levels were significantly increased in serum, milk and amniotic fluid after administration of KCN. In conclusion, the results of the present study evidence that the metabolism of CN varies greatly considering the physiologic state of the female rat, being females at estrus probably more exposed by these substances than at gestation and lactation because in these states there are other compartments, fetus and milk, which may capture these substances, as demonstrated by the V(d) values.

Sulforaphane inhibits CYP1A1 activity and promotes genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro.

Increasing environmental pollution by carcinogens such as some of persistent organic pollutants (POPs) has prompted growing interest in searching for chemopreventive compounds which are readily obtainable. Sulforaphane (SFN) is isolated from cruciferous vegetables and has the potentials to reduce carcinogenesis through various pathways. In this study, we studied the effects of SFN on CYP1A1 activity and genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that SFN inhibited TCDD-induced CYP1A1 activity in H4IIE cells by directly inhibiting CYP1A1 activity, probably through binding to aryl hydrocarbon receptor and/or CYP1A1 revealed by molecular docking. However, SFN promoted TCDD-induced DNA damage in yeast cells and reduced the viability of initiated yeast cells. Besides, it is surprising that SFN also failed to reduce genotoxicity induced by other genotoxic reagents which possess different mechanisms to lead to DNA damage. Currently, it is difficult to predict whether SFN has the potentials to reduce the risk of TCDD based on the conflicting observations in the study. Therefore, further studies should be urgent to reveal the function and mechanism of SFN in the stress of such POPs on human health.

Effects of exogenous thiocyanate on mineral nutrients, antioxidative responses and free amino acids in rice seedlings.

The effects of exogenous thiocyanate (SCN(-)) on amino acids composition, content of mineral nutrients and antioxidative systems in plants were investigated. Young rice seedlings (Oryza sativa L. cv. XZX 45) were grown in nutrient solutions amended with potassium thiocyanate (KSCN). Activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in plant materials were analyzed in vivo. Mineral nutrients and free amino acids in rice seedlings were also measured to determine metabolic responses to SCN(-) exposure. A significant reduction in transpiration and relative growth was recorded with all treatments (p < 0.05), while changes of total chlorophyll content in leaves was negligible (p > 0.05). SCN-induced toxicity appeared to be more sensitive to activities of POD in shoots and APX activities in roots than the others. The content of nutrient elements in rice seedlings exposed to exogenous SCN(-) was variable, while the effects were more evident at the highest SCN-treatment (p < 0.05). Although the change of total free amino acids in shoots of SCN-exposed seedlings was negligible (p > 0.05), responses of different amino acids to SCN(-) application were quite different. Among fifteen free amino acids detected, serine (Ser), proline (Pro), and methionine (Met) increased, while asparagine (Asp) decreased with an increase of the doses of SCN(-) supplied. Phyto-transport of SCN(-) was apparent and the removal rates were positively correlated to the doses, suggesting that phyto-assimilation of SCN(-) is an enzymatic process through a potentially un-identified degradation pathway.

Conditional pharmacology/toxicology V: ambivalent effects of thiocyanate upon the development and the inhibition of experimental arthritis in rats by aurothiomalate (Myocrysin®) and metallic silver.

This article discusses the bizarre and contrary effects of thiocyanate, the major detoxication product of hydrogen cyanide inhaled from tobacco smoke or liberated from cyanogenic foods, e.g. cassava. Thiocyanate both (1) promotes inflammatory disease in rats and (2) facilitates the anti-inflammatory action of historic metal therapies based on gold (Au) or silver (Ag) in three models of chronic polyarthritis in rats. Low doses of nanoparticulate metallic silver (NMS) preparations, i.e. zerovalent silver (Ag°) administered orally, suppressed the mycobacterial ('adjuvant')-induced arthritis (MIA) in rats. Similar doses of cationic silver, Ag(I), administered orally as silver oxide or soluble silver salts were inactive. By contrast, NMS only inhibited the development of the collagen-induced arthritis (CIA) and pristane-induced arthritis (PIA) in rats when thiocyanate was also co-administered in drinking water. These (a) arthritis-selective and (b) thiocyanate-inducible effects of Ag° were also observed in some previous, and now extended, studies with the classic anti-arthritic drug, sodium aurothiomalate (ATM, Myocrisin(®)) and its silver analogue (STM), administered subcutaneously to rats developing the same three forms of polyarthritis. In the absence of either Ag° or ATM, thiocyanate considerably increased the severity of the MIA, CIA and PIA, i.e. acting as a pro-pathogen. Hitherto, thiocyanate was considered relatively harmless. This may not be true in rats/people with immuno-inflammatory stress and concomitant leukocyte activation. Collectively, these findings show how the drug action of a xenobiotic might be determined by the nature (and severity) of the experimental inflammation, as an example of conditional pharmacology. They also suggest that an incipient toxicity, even of normobiotics such as thiocyanate, might likewise be modulated beneficially by well-chosen xenobiotics (drugs, nutritional supplements, etc.), i.e. conditional toxicology (Powanda 1995). Thus, both the disease and the environment may determine (1) the therapeutic action and/or (2) adverse effect(s) of xenobiotics--and even some normobiotics.

Transcriptome reveals the crucial role of exogenous hydrogen sulfide in alleviation of thiocyanate (SCN-) toxicity in rice seedlings.

Application of exogenous hydrogen sulfide (H2S) is a novel strategy for alleviation of the adverse effects caused by abiotic stresses. However, little is known about H2S-mediated global molecular response of rice seedlings to thiocyanate (SCN-) exposure. Herein, a hydroponic experiment was carried out to investigate the crucial role of exogenous H2S in alleviation of SCN- toxicity generated at different effective concentrations (EC20: 24.0 mg SCN/L, EC50: 96.0 mg SCN/L, and EC75: 300.0 mg SCN/L) in rice seedlings through transcriptome analysis. The results showed that the total numbers of differentially expressed genes (DEGs, upregulated genes/downregulated genes) in rice roots were 755/313, 1114/3303, and 2184/7427, while they were 427/292, 2134/526, and 2378/890 in rice shoots at EC20, EC50, and EC75 of SCN-, respectively. When exogenous H2S was supplied to rice seedlings exposed to SCN-, the total number of DEGs (upregulated genes/downregulated genes) in rice roots was 1158/316, 1943/2959, and 1737/5392, while it was 2067/937, 2689/683, and 3492/1062 in rice shoots at EC20, EC50, and EC75 of SCN-, respectively. Upregulated DEGs in shoots were positively correlated with SCN- concentration in the presence of exogenous H2S, suggesting its crucial role in regulating the phytotoxicity of SCN-. Gene function and pathway enrichment analyses showed that exogenous H2S triggered "secondary metabolite synthesis," "metabolic pathways," and "signal transduction mechanisms" in rice seedlings corresponding to different effective concentrations of SCN- exposure.

A newly developed consortium with a highly efficient thiocyanate degradation capacity: A comprehensive investigation of the degradation and detoxification potential.

Thiocyanate-containing wastewater harms ecosystems and can cause serious damage to animals and plants, so it is urgent to treat it effectively. In this study, a new efficient thiocyanate-degrading consortium was developed and its degradation characteristics were studied. It was found that up to 154.64 mM thiocyanate could be completely degraded by this consortium over 6 days of incubation, with a maximum degradation rate of 1.53 mM h-1. High-throughput sequencing analysis showed that Thiobacillus (77.78%) was the predominant thiocyanate-degrading bacterial genus. Plant toxicology tests showed that the germination index of mung bean and rice seeds cultured with media obtained after thiocyanate degradation by the consortium increased by 94% and 84.83%, respectively, compared with the control group without thiocyanate degradation. Cytotoxicity tests showed that thiocyanate without degradation significantly decreased the Neuro-2a cell activity and mitochondrial membrane potential; induced reactive oxygen species generation and apoptosis; increased the cellular Ca2+ concentration; and damaged the cell nucleus and DNA. Furthermore, the thiocyanate degradation products produced the consortium were almost totally non-toxic, revealing the same characteristics as those of the control using distilled water. This study shows that the consortium has a high degradation efficiency and detoxification characteristics, as well as great application potential in bioremediation of industrial thiocyanate-containing wastewater.

Hypothiocyanous acid: benign or deadly?

Hypothiocyanous acid (HOSCN) is produced in biological systems by the peroxidase-catalyzed reaction of thiocyanate (SCN(-)) with H(2)O(2). This oxidant plays an important role in the human immune system, owing to its potent bacteriostatic properties. Significant amounts of HOSCN are also formed by immune cells under inflammatory conditions, yet the reactivity of this oxidant with host tissue is poorly characterized. Traditionally, HOSCN has been viewed as a mild oxidant, which is innocuous to mammalian cells. Indeed, recent studies show that the presence of SCN(-) in airways has a protective function, by preventing the formation of other, more damaging, inflammatory oxidants. However, there is an increasing body of evidence that challenges this dogma, showing that the selectivity of HOSCN for specific thiol-containing cellular targets results in the initiation of significant cellular damage. This propensity to induce cellular dysfunction is gaining considerable interest, particularly in the cardiovascular field, as smokers have elevated plasma SCN(-), the precursor for HOSCN. This review will outline the beneficial and detrimental aspects of HOSCN formation in biological systems.

Allylic thiocyanates as a new class of antitubercular agents.

TB is a global public health emergency in which new drugs are desperately needed. Herein we report on the synthesis of a diverse panel of 41 aryl allylic azides, thiocyanates, isothiouronium salts, and N,N'-diacetylisothioureas that were evaluated for their in vitro activity against replicating and non-replicating Mycobacterium tuberculosis (Mtb) H(37)Rv and toxicity to VERO cells. We found a selective group of new and promising compounds having good (micromolar) to excellent (sub-micromolar) potency against replicating Mtb H(37)Rv. Allylic thiocyanates bearing halophenyl (halo=2-Br, 4-Br, 4-Cl, 4-F), 4-methylphenyl and 2-naphthyl moieties were the most active as antitubercular agents. In particular, the 2-bromophenyl-substituted thiocyanate showed MIC=0.25 µM against replicating Mtb, MIC=8.0 µM against non-replicating Mtb and IC(50)=32 µM in the VERO cellular toxicity assay.

Phytotoxicity of thiocyanate to rice seedlings.

The acute toxicity of potassium thiocyanate (KSCN) and ammonium thiocyanate (NH(4)SCN) to rice seedlings was tested. Hydroponically-grown plants showed different responses to the two species of thiocyanate. NH(4)SCN caused more severe stress to rice seedlings than KSCN. A significant reduction in transpiration and relative growth was observed with all NH(4)SCN treatments (p < 0.01), while the effect of KSCN on rice seedlings was more evident at greater than 100 mg SCN/L (p < 0.01). Both chemicals had a negligible effect on total chlorophyll content in shoots of rice seedlings (p > 0.05). Although phyto-transport of thiocyanate was apparent, rice seedlings showed significantly higher removal potential for NH(4)SCN than KSCN.

Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma.

BACKGROUND: Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. DESIGN AND METHODS: We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. RESULTS: We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients' myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G(2)/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/ß and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. CONCLUSIONS: Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.

Role of alpha class glutathione transferases (GSTs) in chemoprevention: GSTA1 and A4 overexpressing human leukemia (HL60) cells resist sulforaphane and curcumin induced toxicity.

Alpha-class glutathione transferases (α-GSTs) have been shown to protect cells from the harmful effects of reactive oxygen species (ROS) induced lipid peroxidation (LPO) during oxidative stress caused by various physico-chemical agents. While GSTA1-1/A2-2 isozymes exhibit high activity towards lipid and fatty acid hydroperoxides through their selenium independent glutathione peroxidase (GPx) activity, the GSTA4-4 isozyme efficiently metabolizes the LPO product 4-hydroxynonenal (4-HNE) by conjugating it with glutathione (GSH). Because of the fact that ROS generated by the chemopreventive agents, sulforaphane (SFN) and curcumin (Cur), are implicated in the mechanisms of cancer cell killing, the present studies were designed to investigate the contribution of ROS induced LPO in the cytotoxic effects of these agents and the role of α-class GSTs in modulating their toxicity. Human erythroleukemic (HL60) cells were stably transfected with the cDNA encoding the hGSTA1-1 and mGsta4-4 isozymes. After analysing the expression and activities of the respective GST isozymes, the effects of SFN and Cur on the extent of LPO, cytotoxicity and apoptosis were compared in empty vector (VT), hGSTA1-1 and mGsta4-4 expressing HL60 cells. These studies demonstrate that when compared with SFN, Cur was relatively more cytotoxic to HL60 cells. The ectopic expression of hGSTA1-1 and mGsta4-4 isozymes provided resistance to SFN and Cur induced cytotoxicity and apoptosis through a significant suppression of LPO in these cells. Overall, the results suggest that the expression of α-class GSTs in cancer cells can modulate the therapeutic efficacy of chemopreventive agents.

Fuzzy synthetic evaluation of the impact of plant growth regulators on the root phenotype traits of rice seedlings under thiocyanate stress.

Application of plant growth regulators (PGRs) is a novel strategy for allay of the adverse effects caused by biotic/abiotic stresses. However, no studies have vividly executed mathematic evaluation for the assessment of various PGRs on root phenotype traits (RPTs) against pollutants. In the present study, a microcosm hydroponic experiment was conducted to examine responses of RPTs under SCN- (0, 24, 96, and 300 mg SCN/L) stress in the presence of PGRs such as jasmonic acid (JA), indole-3-acetic acid (IAA), and sodium hydrosulfide (NaHS) in rice plants. Fuzzy synthetic evaluation was applied to determine the outcome of the effects of various PGRs on the RPTs under SCN- exposure. Root scanning results indicated that exogenous IAA and NaHS has the greater potential for improving the RPTs of rice seedlings under SCN- stress, while JA failed to uplift the RPTs in response to SCN- stress. Fuzzy synthetic evaluation indicated that in control plants (without SCN-), the effect of three PGRs applied on the RPTs is as follows: NaHS > IAA > JA. At 24 mg SCN/L, NaHS and IAA had consistent actuate in regulating RPTs of rice seedlings, while all PGRs amended have an affirmative impact on RPTs at 96 and 300 mg SCN/L. The present research highlights the utilization of contemporary mathematic method to screen the superior species of PGRs through the RPTs test of plants under pollutant belt.

Sulforaphane induces DNA single strand breaks in cultured human cells.

Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 microM SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value of SFR in anticancer drug protocols.