STAT3 Pathways Contribute to ß-HCH Interference with Anticancer Tyrosine Kinase Inhibitors.

Organochlorine pesticides (OCPs) are a class of environmentally persistent and bioaccumulative pollutants. Among these, ß-hexachlorocyclohexane (ß-HCH) is a byproduct of lindane synthesis, one of the most worldwide widespread pesticides. ß-HCH cellular mechanisms inducing chemical carcinogenesis correspond to many of those inducing chemoresistance, in particular, by the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathways. For this purpose, four cell lines, representative of breast, lung, prostate, and hepatocellular cancers, were treated with ß-HCH, specific tyrosine kinase inhibitors (TKIs), and a STAT3 inhibitor. All cell samples were analyzed by a viability assay, immunoblotting analysis, a wound-healing assay, and a colony formation assay. The results show that ß-HCH reduces the efficacy of TKIs. The STAT3 protein, in this context, plays a central role. In fact, by inhibiting its activity, the efficacy of the anticancer drug is restored. Furthermore, this manuscript aimed to draw the attention of the scientific and socio-healthcare community to the issue of prolonged exposure to contaminants and their impact on drug efficacy.

Status of Acaricide Resistance and Detecting the Knockdown Resistance Mutation T2134A in the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae) from Northeastern Mexico.

Rhipicephalus microplus is the most important tick in veterinary medicine, given its repercussions on animal production. The principal strategy to avoid adverse effects associated with R. microplus is the chemical control of tick populations through organosynthetic acaricides. Therefore, monitoring susceptibility to acaricides is paramount in any control program. This study aimed to analyze the resistance status of 2 populations of R. microplus from northeastern Mexico to the organochlorine (OC) lindane, organophosphates (OP) coumaphos, chlorfenvinphos, diazinon, and chlorpyrifos, and the synthetic pyrethroids (SPs) flumethrin, deltamethrin, and cypermethrin. Discriminating doses (DD) of each acaricide were used in the larval packet bioassay (LPT). Additionally, the presence of the knockdown resistance (kdr) mutation T2134A associated with pyrethroid resistance was evaluated using allele-specific polymerase chain reaction (PCR). The populations of R. microplus showed a high frequency of resistance to SP, with mortality rates of less than 5%; they also showed resistance to the OPs (diazinon and chlorpyrifos) with mortality rates ranging from 1.29% to 34.62%; meanwhile, they were susceptible to coumaphos and chlorfenvinphos. Mortality rates higher than 66% were observed for lindane, indicating susceptibility. The mutant allele of the kdr mutation T2134A was detected in 75% and 100% of the pools analyzed. The populations studied presented a highly resistant profile to pyrethroids, with the presence of the kdr mutant allele A2134. The susceptibility to the organophosphates such as coumaphos and chlorfenvinphos of R. microplus from northeastern Mexico should be noted.

ß-Hexachlorocyclohexane Drives Carcinogenesis in the Human Normal Bronchial Epithelium Cell Line BEAS-2B.

Organochlorine pesticides constitute the majority of the total environmental pollutants, and a wide range of compounds have been found to be carcinogenic to humans. Among all, growing interest has been focused on ß-hexachlorocyclohexane (ß-HCH), virtually the most hazardous and, at the same time, the most poorly investigated member of the hexachlorocyclohexane family. Considering the multifaceted biochemical activities of ß-HCH, already established in our previous studies, the aim of this work is to assess whether ß-HCH could also trigger cellular malignant transformation toward cancer development. For this purpose, experiments were performed on the human normal bronchial epithelium cell line BEAS-2B exposed to 10 µM ß-HCH. The obtained results strongly support the carcinogenic potential of ß-HCH, which is achieved through both non-genotoxic (activation of oncogenic signaling pathways and proliferative activity) and indirect genotoxic (ROS production and DNA damage) mechanisms that significantly affect cellular macroscopic characteristics and functions such as cell morphology, cell cycle profile, and apoptosis. Taking all these elements into account, the presented study provides important elements to further characterize ß-HCH, which appears to be a full-fledged carcinogenic agent.

Precise control of ion channel and gap junction expression is required for patterning of the regenerating axolotl limb.

Axolotls and other salamanders have the capacity to regenerate lost tissue after an amputation or injury. Growth and morphogenesis are coordinated within cell groups in many contexts by the interplay of transcriptional networks and biophysical properties such as ion flows and voltage gradients. It is not, however, known whether regulators of a cell's ionic state are involved in limb patterning at later stages of regeneration. Here we manipulated expression and activities of ion channels and gap junctions in vivo, in axolotl limb blastema cells. Limb amputations followed by retroviral infections were performed to drive expression of a human gap junction protein Connexin 26 (Cx26), potassium (Kir2.1-Y242F and Kv1.5) and sodium (NeoNav1.5) ion channel proteins along with EGFP control. Skeletal preparation revealed that overexpressing Cx26 caused syndactyly, while overexpression of ion channel proteins resulted in digit loss and structural abnormalities compared to EGFP expressing control limbs. Additionally, we showed that exposing limbs to the gap junction inhibitor lindane during the regeneration process caused digit loss. Our data reveal that manipulating native ion channel and gap junction function in blastema cells results in patterning defects involving the number and structure of the regenerated digits. Gap junctions and ion channels have been shown to mediate ion flows that control the endogenous voltage gradients which are tightly associated with the regulation of gene expression, cell cycle progression, migration, and other cellular behaviors. Therefore, we postulate that mis-expression of these channels may have disturbed this regulation causing uncoordinated cell behavior which results in morphological defects.

Assembly of root-associated microbiomes of typical rice cultivars in response to lindane pollution.

Organochlorine pesticides have been extensively used for many years to prevent insect diseases of rice (Oryza sativa L.), but little is known about their residual impacts on the underground micro-ecology in anaerobic environment. In this glasshouse study, we characterized the lindane effects on the assembly of root-associated microbiomes of commonly used indica, japonica and hybrid rice cultivars, and their feedback in turn, in modifying lindane anaerobic dissipation during 60 days' rice production. The results showed that rice growth inhibited the anaerobic dissipation of lindane, but was not affected apparently by lindane at initial spiked concentration of 4.62 and 18.54 mg kg-1 soil. Suppressed removal of lindane in rice planted treatments as compared with that in unplanted control was likely due to inhibited reductive dechlorination induced by a comprehensive effect of radial O2 secretion of rice root and co-occurring Fe(III) reduction that consumed electron competitively in rice rhizosphere. However, the hybrid cultivar exhibited a less suppression than the conventional cultivars in high polluted soils. Bacteria was more sensitively responded to lindane pollution than fungal taxa, and Actinobacteria, Chloroflexi, Verrucomicrobia and Proteobacteria were the main different phyla between hybrid and conventional cultivars, with a more stable community structure exhibited in the hybrid rice under lindane stress. Our study highlights the assembly and variation of root-associated microbiomes in responses of lindane pollution, and suggests that hybrid rice cultivar might be most competent for cultivation in paddy fields polluted by lindane and other organochlorine pesticides, especially in the area with high residual levels.

Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex-Mouse Subplate Zone.

Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1-P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 µM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1-P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.

Multi-generational effects of lindane on nematode lipid metabolism with disturbances on insulin-like signal pathway.

Influences on lipid metabolism and multi-generational obesogenic effects raised new concerns on lipophilic pollutants (e.g., lindane). Yet, the mechanisms remained unanswered. The present study exposed Caenorhabditis elegans to lindane for 4 consecutive generations (F0 to F3) at 1.0 ng/L, and measured effects in the directly exposed generations (F0 to F3), indirectly exposed ones (T1 and T1') and un-exposed ones (T3 and T3'). Lindane stimulated fat storages in all generations. At the biochemical level, lindane stimulated both acetyl-CoA carboxylase (ACC) and carnitine palmitoyl-transferases (CPT) in F0, T1 and T2, while inhibited them in F3, T1' and T3', demonstrating the balance between fatty acid synthesis and its depletion toward fat accumulation over generations. Moreover, lindane caused different effects on insulin among generations. It inhibited insulin in F0 and F3 and exhibited consistent effects on the expression changes of daf-2, sgk-1 and daf-16 genes in insulin-like signal pathway. Lindane also inhibited insulin in T1 and T3 but exhibited consistent effects on the expression changes of daf-2, akt-1 and daf-16. Different roles of sgk-1 and akt-1 indicated the response strategies from tolerance (F0 and F3) to avoidance (T1 and T3). Lindane stimulated insulin in T1' and T3' and exhibited consistent effects on expression changes of daf-2, sgk-1 and daf-16 genes that were similar in F0 and F3.

STAT3, a Hub Protein of Cellular Signaling Pathways, Is Triggered by ß-Hexaclorocyclohexane.

BACKGROUND: Organochlorine pesticides (OCPs) are widely distributed in the environment and their toxicity is mostly associated with the molecular mechanisms of endocrine disruption. Among OCPs, particular attention was focused on the effects of ß-hexaclorocyclohexane (ß-HCH), a widely common pollutant. A detailed epidemiological study carried out on exposed population in the "Valle del Sacco" found correlations between the incidence of a wide range of diseases and the occurrence of ß-HCH contamination. Taking into account the pleiotropic role of the protein signal transducer and activator of transcription 3 (STAT3), its function as a hub protein in cellular signaling pathways triggered by ß-HCH was investigated in different cell lines corresponding to tissues that are especially vulnerable to damage by environmental pollutants. MATERIALS AND METHODS: Human prostate cancer (LNCaP), human breast cancer (MCF-7 and MDA-MB 468), and human hepatoma (HepG2) cell lines were treated with 10 µM ß-HCH in the presence or absence of specific inhibitors for different receptors. All samples were subjected to analysis by immunoblotting and RT-qPCR. RESULTS AND CONCLUSIONS: The preliminary results allow us to hypothesize the involvement of STAT3, through both its canonical and non-canonical pathways, in response to ß-HCH. Moreover, we ascertained the role of STAT3 as a master regulator of energy metabolism via the altered expression and localization of HIF-1α and PKM2, respectively, resulting in a Warburg-like effect.

Metabolic synergies in the biotransformation of organic and metallic toxic compounds by a saprotrophic soil fungus.

The saprotrophic fungus Penicillium griseofulvum was chosen as model organism to study responses to a mixture of hexachlorocyclohexane (HCH) isomers (α-HCH, ß-HCH, γ-HCH, δ-HCH) and potentially toxic metals (vanadium, lead) in solid and liquid media. The P. griseofulvum FBL 500 strain was isolated from polluted soil containing high concentrations of HCH isomers and potentially toxic elements (Pb, V). Experiments were performed in order to analyse the tolerance/resistance of this fungus to xenobiotics and to shed further light on fungal potential in inorganic and organic biotransformations. The aim was to examine the ecological and bioremedial potential of this fungus verifying the presence of mechanisms that allow it to transform HCH isomers and metals under different extreme test conditions. To our knowledge, this work is the first to provide evidence on the biotransformation of HCH mixtures, in combination with toxic metals, by a saprotrophic non-white-rot fungus and on the metabolic synergies involved.

Randomized, investigator-blinded, controlled clinical study with lice shampoo (Licener®) versus dimethicone (Jacutin® Pedicul Fluid) for the treatment of infestations with head lice.

The present clinical trial was conducted to obtain additional data for the safety and efficacy of a head lice shampoo that is free of silicone compared with an anti-head lice product containing dimethicone. Both products act by a physical mode of action. This randomized, investigator-blinded, controlled clinical study was conducted between July and November 2016 in households of two villages (Abou Rawash and Shandalat) in Egypt. Children older than 2 years with an active head lice infestation were treated with either a shampoo-based head lice treatment containing neem extract (Licener®) or dimethicone (Jacutin® Pedicul Fluid) on day 1 and additionally on day 9. Assessment for living lice by combing was conducted before and 1-2 h after treatment and on days 5 and 13. The main objective was to demonstrate a cure rate of the test product of at least 85% after a single application (day 5 and 9). Secondary objectives were to scrutinize patient safety and satisfaction as well as cure rates on day 13 after two treatments and the evaluation of ovicidal and licicidal efficacies of the products. Sixty-one children in the test-group (Licener®) and 58 children in the reference group (Jacutin® Pedicul Fluid) were included in this study. The test product and the reference product were very well tolerated. Both products exceeded the objective of cure rates of over 85% after single treatment (test group 60/60 = 100%; 95% CI = 94.04-100.00%; reference group 54/57 = 94.74%; 95% CI = 85.38-98.90%; p = 0.112; CI by Clopper-Pearson) and after two treatments (test group 58/58 = 100%; 95% CI = 93.84-100.00%; reference group 52/54 = 96.30%; 95% CI = 87.25-99.55%; p = 0.230) with higher cure rates and non-inferiority for the test product. The combined success rate shows significant superiority of the test product against the reference product (test group 58/58 = 100%; 95% CI = 93.84-100.00%; reference group 49/54 = 90.7%; 95% CI = 79.70-96.92%; p = 0.024). The test product showed higher ovicidal efficacy than the reference product. Thus, the present study demonstrates that a single treatment with a head lice product like Licener® can be sufficient to eliminate a head lice infestation.

Organochloride pesticides impaired mitochondrial function in hepatocytes and aggravated disorders of fatty acid metabolism.

p,p'-dichlorodiphenyldichloroethylene (p, p'-DDE) and ß-hexachlorocyclohexane (ß-HCH) were two predominant organochlorine pesticides (OCPs) metabolites in human body associated with disorders of fatty acid metabolism. However, the underlying mechanisms have not been fully clarified. In this study, adult male C57BL/6 mice were exposed to low dose of p, p'-DDE and ß-HCH for 8 wk. OCPs accumulation in organs, hepatic fatty acid composition, tricarboxylic acid cycle (TCA) metabolites and other metabolite profiles were analyzed. Expression levels of genes involved in hepatic lipogenesis and ß-oxidation were measured. Mitochondrial function was evaluated in HepG2 cells exposed to OCPs. High accumulation of p, p'-DDE and ß-HCH was found in liver and damaged mitochondria was observed under electron microscopy. Expression of genes in fatty acid synthesis increased and that in mitochondrial fatty acid ß-oxidation decreased in OCPs treatment groups. OCPs changed metabolite profiles in liver tissues, varied hepatic fatty acid compositions and levels of several TCA cycle metabolites. Furthermore, MitoTracker Green fluorescence, ATP levels, mitochondrial membrane potential and OCR decreased in HepG2 cells exposed to OCPs. In conclusion, chronic exposure to OCPs at doses equivalent to internal exposures in humans impaired mitochondrial function, decreased fatty acid ß-oxidation and aggravated disorders of fatty acid metabolism.

Concomitant effect of low dose of lindane and intranasal lipopolysaccharide on respiratory system of mice.

Lindane is very commonly used organochlorine pesticide and has been reported to cause several toxic effects including respiratory insufficiency. However, effects of low concentration of lindane alone or in combination with microbial molecules on lungs are not fully understood. To understand the effects a preliminary study was designed on Swiss albino mouse. Male mice were divided into treatment and control group (20; each). Treatment mice were given lindane in ground nut oil orally at 0.25 mg kg-1 day-1 for 60 days. After treatment, 10 mice were challenged with intranasal Escherichia coli lipopolysaccharide (LPS; 80 µg per mice) and remaining 10 with normal saline. The mice were euthanized 16 h post-LPS exposure. Control mice (10 each) were given normal saline or the LPS alone. Mice exposed with lindane and in combination with LPS had increase in total cell counts and leukocyte counts in broncho-alveolar lavage. Histological examination showed lung injury in the lindane-treated mice. The histopathological changes were more pronounced in lindane along with LPS-exposed mice. Lindane alone and in combination with LPS showed expression of immunopositive Toll-like receptor (TLR)-4 and tumour necrosis factor-alpha (TNF-α) positive reaction in various cells of lungs. While LPS induced acute inflammation in the lungs, combination of lindane and LPS exacerbated histological signs of the inflammation. The data indicate that lindane alone or in combination with LPS caused changes in lung morphology and altered TLR-4 and TNF-α expression which may have led to altered response to LPS challenge.

Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line.

The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1µM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50=116nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening.

The "adaptive responses" of low concentrations of HBCD in L02 cells and the underlying molecular mechanisms.

This study aimed to investigate the "adaptive responses" of hexabromocyclododecanes (HBCD) at environmentally relevant concentrations in human hepatocytes L02. L02 cells were pre-treated with low concentrations of HBCD (10(-13)-10(-11) M), followed by treatment with high concentrations of HBCD, α-hexachlorocyclohexane (α-HCH), polychlorinated biphenyls (PCBs), or polybrominated diphenyl ether-47 (BDE47). The results showed that the pre-treatment with low concentrations of HBCD induced "adaptive responses" to high concentrations of HBCD/α-HCH exposure (but not to PCBs and BDE47), as evidenced by attenuation of survival inhibition, reactive oxygen species (ROS) over-production, and deoxyribonucleic acid (DNA) damage induction. The "adaptive responses" induced by low concentrations of HBCD, which depended on the activation of the phosphatidylinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, reduced the phosphorylation of adenosine monophosphate-activated kinase (AMPK) and enhanced the phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). The observations were further confirmed by the experiments with inhibitors. Moreover, the evaluation on the changes of metabolic enzymes revealed that HBCD and α-HCH shared a similar pattern of cytochrome P450 induction (CYP2B6), which was different from those of PCBs and BDE47 (CYP1A1 and CYP2B6). These results indicated that low concentrations of HBCD could induce "adaptive responses" to the subsequent treatment with high concentrations of HBCD/α-HCH in L02 cells, which was associated with the PI3K/Akt pathway, and AMPK and p38 MAPK signaling. The "adaptive responses" seemed to be dependent on the types of chemicals in terms of the metabolic patterns and chemical structures.

γ-Lindane Increases Microcystin Synthesis in Microcystis aeruginosa PCC7806.

HCH factories, and the waste dumpsites associated to its production, have become a global environmental concern, and their runoff could pollute ground and surface waters with high levels of the pollutant. In this study, the influence of lindane (γ-HCH) on microcystin production has been investigated in Microcystis aeruginosa PCC7806. This toxic cyanobacterium is highly tolerant to γ-lindane (20 mg/L), and produces more toxin (microcystin) in the presence of the pollutant. Microcystis degrades γ-lindane and presence of γ-lindane induces genes involved in its own degradation (nirA). RT-PCRsq has been used to monitor changes in levels of transcripts encoded by the mcy operon (mcyD, mcyH and mcyJ), responsible for the microcystin synthesis machinery, as well as other genes involved in its transcriptional regulation, such as ntcA and fur family members. The presence of lindane in the culture media induces mcyD expression, as well as ntcA gene transcription, while other genes, such as mcyH, (putative ABC transporter), are downregulated. The amount of microcystin found in the cells and the culture media is higher when M. aeruginosa is treated with γ-lindane than in control cells. The results suggest that in a lindane polluted environment, Microcystis toxic strains may enhance their microcystin synthesis.

γ-BHC: Its history and mystery--why is only γ-BHC insecticidal?

Among BHC isomers (benzene hexachloride, C6H6Cl6, theoretically eight ones), seven isomers (α,ß,γ,δ,ε,η,ι) including one racemate (α) were isolated and their configurations were elucidated. Among the seven isomers, only γ-BHC has a potent insecticidal activity. γ-BHC poisoning symptoms accompanied by the violent tremor of the body, particularly in the legs and abnormal fluttering were consistently correlated with central nervous system effects, and the action was shown on the ganglia rather than on the isolated nerve cord. The excitatory effects of γ-BHC poisoning on spontaneous activity and synaptic function in the sixth abdominal ganglion of the American cockroach result from the presynaptic action that causes an excessive release of acetylcholine. F. Matsumura found the phenomena of cross resistance between γ-BHC, cyclodiene insecticides and GABA (γ-aminobutyric acid) antagonist, picrotoxinin, and concluded that the primary target of γ-BHC is the picrotoxinin receptor in the GABA receptor Cl- ion channel complex (GABA-gated chloride channel) and its antagonistic action on GABA receptor results in an excessive release of Ach. In order to study the structure-activity relationship, a number of γ-BHC analogs, in which one or two chlorine atoms on dl or meso position(s) of the γ-BHC molecule were replaced by various substituents such as hydrogen, halogens other than chlorine and alkoxy groups, etc. were synthesized. In the case of dl type-analogs, there is an optimum volume for dl-substituents, which corresponds to Cl. The question why γ-BHC is insecticidal and other BHC isomers are not remains unsolved.

Cross-resistance between fipronil and lindane in Rhipicephalus (Boophilus) microplus.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is one of the most damaging parasites of cattle in tropical and subtropical regions. Several chemical groups have been used for its control, including cyclodienes (lindane and dieldrin). In Uruguay and Brazil these products were used at the beginning of the 1960s and during a few years. Fipronil and lindane act on the same target site. In both countries, southern cattle tick resistance to fipronil has sometimes developed quickly after only a few acaricide treatments (three to seven). The objective of the present study was to determine cross-resistance between fipronil and lindane in southern cattle ticks from Uruguay and Brazil. Initially, the FAO's (Food and Agricultural Organization) larval packet test with lindane was applied to a fipronil-resistant strain and to susceptible field populations. Mozo and POA strains were used as the susceptible controls. A larval immersion test was used to assess fipronil toxicity. Of fifteen fipronil-resistant field populations that were tested with lindane, eleven were lindane-resistant and three were susceptible. The last three populations had incipient resistance to fipronil. Finally, cross-resistance between fipronil and lindane in the southern cattle tick is reported in this study for the first time.

Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations.

Glycine receptor (GlyR) chloride channels mediate fast inhibitory neurotransmission in the spinal cord and brainstem. Four GlyR subunits (α1-3, ß) have been identified in humans, and their differential anatomical distributions underlie a diversity of synaptic isoforms with unique physiological and pharmacological properties. To improve our understanding of these properties, we induced the formation of recombinant synapses between cultured spinal neurons and HEK293 cells expressing GlyR subunits of interest plus the synapse-promoting molecule, neuroligin-2A. In the heterosynapses thus formed, recombinant α1ß and α3ß GlyRs mediated fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2ß GlyRs mediated slow decaying IPSCs. These results are consistent with the fragmentary information available from native synapses and single channel kinetic studies. As ß subunit incorporation is considered essential for localizing GlyRs at the synapse, we were surprised that α1-3 homomers supported robust IPSCs with ß subunit incorporation accelerating IPSC rise and decay times in α2ß and α3ß heteromers only. Finally, heterosynapses incorporating α1(D80A)ß and α1(A52S)ß GlyRs exhibited accelerated IPSC decay rates closely resembling those recorded in native synapses from mutant mice homozygous for these mutations, providing an additional validation of our technique. Glycinergic heterosynapses should prove useful for evaluating the effects of drugs, hereditary disease mutations or other interventions on defined GlyR subunit combinations under realistic synaptic activation conditions.

Influence of soy isoflavone on lindane cumulant in sprague-dawley rats.

Female Sprague-Dawley rats weighing 60-80 g were given different dosages of soy isoflavones and/or lindane for four weeks. Soy isoflavones was added in feed and lindane was given by oral gavage. We found that soy isoflavones could reduce the level of lindane in rat's serum and brain, but might cause the uterus hyperplasia. Lindane could inhibit the effect of soy isoflavones on uterus and significantly decrease the level of estradiol and testosterone in serum. This study indicated that soy isoflavones could reduce the level of lindane in rat's body. Lindane could reduce the level of hormones and decreased the effect of soy isoflavones on rat's uterus.

Effect of treatment of cow's urine "Gomutra" and antioxidants in alleviating the lindane-induced oxidative stress in kidney of Swiss mice (Mus musculus).

The study aimed to evaluate the effect of cow urine and combination of antioxidants against lindane induced oxidative stress in Swiss mice. Male healthy mice, 8-10 weeks old, weighing 30 ± 5 g were randomly selected and divided into eight groups, namely, control (C); lindane (L); antioxidant (A), antioxidant+lindane (A+L), cow urine (U), cow urine+lindane (U+L), cow urine+antioxidants (U+A) and cow urine+antioxidants+lindane (U+A+L). Group C animals were administered only the vehicle (olive oil); doses selected for other treatments were: lindane: 40 mg/kg b.w.; antioxidants: 125 mg/kg b.w. (vitamin C: 50 mg/kg b.w., vitamin E: 50 mg/kg b.w., α-lipoic acid: 25 mg/kg b.w.) and cow urine: 0.25 ml/kg b.w. In group A+L and U+L antioxidants and cow urine were administered 1 h prior to lindane administration and in group U+A and U+A+L cow urine was administered 10 min before antioxidants. All treatments were administered orally continuously for 60 days. Lindane treated group showed increased lipid peroxidation, whereas glutathione, glutathione peroxidase, superoxide dismutase, catalase, protein and endogenous levels of vitamin C and E were significantly decreased compared to control. Administration of cow urine and antioxidants alleviated the levels of these biochemical parameters.