In vitro exposure of human spermatozoa to bisphenol A induces pro-oxidative/apoptotic mitochondrial dysfunction

As bisphenol A (BPA) exerts oxidative/pro-apoptotic effects in several cell types, we explored whether the in vitro exposure to BPA could affect human sperm integrity through the induction of pro-oxidative/apoptotic mitochondrial dysfunction. The exposure of motile sperm suspensions to scalar BPA concentrations for 4 h produced a decrease in the mitochondrial membrane potential, starting from 300 μM. It was associated with an increased mitochondrial generation of superoxide anion, caspase-9 and caspase-3 activation and motility decrement. Vitality decline was observed at BPA ≥ 400 μM. Twenty hours exposure to 300 μM BPA, but not to lower concentrations, produced a significant loss in sperm vitality associated with a complete sperm immobilization. Finally, 300 μM BPA also produced a significant DNA oxidative damage, as revealed by the formation of oxidized base adduct 8-hydroxy-2′-deoxyguanosine. In conclusion, BPA affected human sperm integrity by inducing pro-oxidative/apoptotic mitochondrial dysfunction.     

Oleanolic acid and ursolic acid induce apoptosis in four human liver cancer cell lines

Apoptotic effects of oleanolic acid (OA) and ursolic acid (UA) on human liver cancer HepG2, Hep3B, Huh7 and HA22T cell lines were examined. OA or UA at 2, 4, 8 μmol/L were used and their effects on cell viability, DNA fragmentation, mitochondrial membrane potential (MMP), activity of Na⁺–K⁺-ATPase, caspase-3 and caspase-8, cell adhesion, level of intercellular adhesion molecule (ICAM)-1 and vascular endothelial growth factor (VEGF) in these cell lines were determined. OA or UA treatments concentration-dependently decreased cell viability and increased DNA fragmentation in HepG2 and Hep3B cell lines (P < 0.05). However, these two compounds reduced viability and increased DNA fragmentation in Huh7 cell only at 4 and 8 μmol/L (P < 0.05). OA or UA treatments concentration-dependently lowered MMP in HepG2, Hep3B and HA22T cell lines (P < 0.05). These two compounds also concentration-dependently diminished Na⁺–K⁺-ATPase activity and VEGF level in four test cell lines (P < 0.05). Besides Huh7 cell, OA or UA treatments concentration-dependently elevated caspase-3 and caspase-8 activities in other three cell lines (P < 0.05). Besides HA22T cell, these two compounds concentration-dependently inhibited cell adhesion and decreased ICAM-1 level in other three cell lines (P < 0.05). These findings support that OA and UA are potent anti-cancer agents to cause apoptosis in these liver cancer cell lines.     

Effect of Quercetin-loaded liposomes on induced oxidative stress in human spermatozoa

A strategy to circumvent the poor polyphenols bioavailability is to load these compounds into liposomes. We evaluated the in vitro effects of quercetin (Q) and Q-loaded liposomes (QLL, 30, 50, 100 μM) on motility, viability and chromatin integrity of swim-up selected human sperm. Antioxidant power was assayed against tert-butylhydroperoxide induced lipid peroxidation (LPO) using C11-BODIPY581/591 fluorescent probe and transmission electron microscopy. QLL showed decreased toxicity for sperm motility and viability and increased DNA damage compared to Q. The percentage of sperm with fluorescence, marker of LPO, was decreased in samples incubated with Q vs QLL (P < 0.001). The ultrastructure of acrosomes and membranes was preserved with Q 30/100 μM, whereas QLL did not prevent membrane injury.Q alone appeared more effective than Q incorporated into liposomes; however liposomes could be considered as carriers that may convey different compounds inside sperm; they may therefore represent a field of research rich of many applications.     

Further preliminary assessment of three human glioma cell lines as models of human astrocytic toxicity in vitro

Three human astroglioma lines U251-MG, U373-MG and CCF-STTG1 have been evaluated further as possible models for astrocytotoxicity (GFAP and IL-6 release). The effects of bacterial lipopolysaccharide, chloroquine diphosphate and acrylamide were studied on GFAP expression and LPS, chloroquine diphosphate, ethanol, trimethyltin chloride (TMTC) and acrylamide were examined on interleukin-6 (IL-6) release in the U373-MG line only. At 4-h LPS elevated GFAP (17.0 ± 5.0% P < 0.05) above control in the U251-MG cell line only. Chloroquine diphosphate over 4 h in the U251-MG line resulted in an increase in GFAP-IR to 20.3 ± 4.2% and 21.1 ± 4.1% above control levels 0.1 μM (P < 0.05) and 1 μM (P < 0.05) respectively. CQD was associated with decreases in MTT turnover, particularly after 24 h incubation. With the U373-MG line, LPS (0.5 μg/ml) increased IL-6 expression 640% above control (P < 0.001), whilst chloroquine diphosphate (100 μM), ethanol (10 mM) and TMTC chloride (1 μM) also increased IL-6. It is possible that batteries of astrocytic human glioma cell lines may be applicable to the sensitive evaluation of toxicants on astrogliotic expression markers such as GFAP and IL-6.     

Zearalenone inhibits testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling and orphan nuclear receptor Nur77 expression

Zearalenone (ZEA) directly inhibits testosterone biosynthesis in Leydig cells, although the mechanisms involved remains unclear. Various experiments were performed to elucidate the molecular pathway of ZEA-mediated androgen inhibition. Leydig cells were isolated from 6 week-old male ICR mice and subjected to ZEA pre-treatment. The levels of testosterone and a series of influncing factors were measured. The results showed that ZEA caused a concentration- and time-dependent inhibition of testosterone stimulated both by hCG and cAMP (P < 0.05). Exposure to ZEA did not affect the LHR binding activity nor the protein expression (P > 0.05). However, ZEA exposure significantly elevated the cellular cAMP levels (P < 0.05) in low concentrations (5 μg/ml) or for long time periods (24 h), significantly reduce the mitochondrial membrane potential (P < 0.05). The expression of P450scc, 17β-HSD, and P450c17 at the mRNA level were significantly decreased (P < 0.05). The steroidogenic acute regulatory (StAR) and 3β-HSD expression was significantly increased (P < 0.05). Furthermore, the ERα protein expression was not affected by ZEA, but Nur77 expression was significantly inhibited (P < 0.05). These observations imply that ZEA activity interferes with testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling and Nur77 expression.     

Curcumin and β-caryophellene attenuate cadmium quantum dots induced oxidative stress and lethality in Caenorhabditis elegans model system

Curcumin (CUR) and β-caryophellene (BCP) are well known bioactive phytomolecules which are known to reduce oxidative stress in living organisms. Therefore, the present study was envisaged to explore the possible effects of CUR and BCP in suppression of cadmium quantum dots (CdTe QDs) induced toxicity in Caenorhabditis elegans. CdTe QD are luminescent nanoparticles extensively exploited for in vivo imaging, but long term bioaccumulation confer deleterious effects on living organisms. The 24-h LC₅₀ and LC₁₀₀ of CdTe QD were found to be 18.40 μg/ml and 100 μg/ml respectively. The CdTe QD exposure elevated HSP-16.2 expression mediating induction of the stress response. The CdTe QD lethality was due to increment in ROS and decline in SOD and GST expression. The present study demonstrates improved survival in BCP (50 μM) and CUR (20 μM) treated worms by over 60% (P < 0.01) and 50% (P < 0.029) in CdTe QD (100 μg/ml) exposed worms. Furthermore, BCP and CUR attenuate oxidative stress triggered by QD. The present study for the first time demonstrates CdTe QD toxicity remediation via BCP and CUR. The future investigations can unravel underlying protective effects of phytomolceules for remediating cyotoxicolgical effects of QDs.     

Greater efficacy of the ECMS-oil adjuvant over other formulations on immune responses against Bordetella bronchiseptica in rabbits and the underlying mechanism

In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS + oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800 μg + oil group were significantly higher than in the other adjuvant groups (p < 0.05) on day 21. The agglutination antibody titer was also higher than the other groups (p < 0.05) on day 37. The ECMS800 μg + oil group improved cellular immune responses compared to other adjuvant groups, including control using antigen alone without adjuvant and the PBS group (p < 0.05). After Bb challenge, the ECMS800 μg + oil group showed the highest protection rate, which was significantly higher than ECMS alone or control using antigen alone without adjuvant and the PBS group (p < 0.05 and p < 0.01). IgA cells in the ECMS800 μg + oil group differed significantly from the IgA cells of other groups in the lungs (p < 0.01). The results of cell recruitment showed that the number of lymphocytes in the ECMS400 μg + oil were higher than the number of cells for other groups except the ECMS(100 μg/800 μg) + oil groups (p < 0.05). Intermediate cells in the ECMS(100 μg/400 μg) + oil groups were higher than the number of cells for other groups, including the control using antigen alone group (p < 0.05). Neutrophils in the ECMS(100 μg/400 μg/800 μg) + oil groups were significantly higher than the ECMS 800 μg and control using antigen alone groups (p < 0.05). White blood cells in the ECMS100 μg + oil group were significantly higher than the oil, ECMS800 μg and control using antigen alone groups (p < 0.05). IL-2 expression in the ECMS800 μg + oil group was significantly higher than other groups, except for the ECMS400 μg + oil group (p < 0.05). IL-4 expression in the ECMS800 μg + oil group was significantly higher than other groups (p < 0.05). GATA3 in the ECMS800 μg + oil groups was significantly higher than the oil, ECMS800 μg and control using antigen alone group (p < 0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits.     

Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa

Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4 μmol/mL following incubation for 1.5 h. Notably, all parameters were potently inhibited by 10 μmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of Cr (>10 μmol/mL) increased the tyrosine phosphorylation of some high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.     

Identification of pathology from diesel exhaust particles in the bladder in a rat model by aspiration of particles from the pharynx

To determine whether diesel exhaust particles (DEPs) could be a toxic agent to the bladder, rats were exposed to different concentrations of DEPs for one month or three months. When the rats were sacrificed, morphologic changes of the urothelium were investigated. The antioxidase activity and the levels of lipid peroxidation in the bladder were assayed. In the three-month group, DEPs at doses of 21.03 μg/μl insulted the structural integrity of surface glycosaminoglycans, widened the gap between urothelial cells, increased levels of lipid peroxidation, and decreased antioxidase activities in the urinary bladder (p < 0.05). Furthermore, DEPs at a dose of 5.61 μg/μl decreased glutathione, catalase, and glutathione peroxidase activities (p < 0.05). These results led to the conclusion that DEPs were a toxic agent in the bladder. The toxic effects might be attributed to oxidative damage mediated by pro-oxidant/antioxidant imbalance or excessive free radicals.     

Ethyl acetate fraction of Garcina epunctata induces apoptosis in human promyelocytic cells (HL-60) through the ROS generation and G0/G1 cell cycle arrest: A bioassay-guided approach

Number of deaths due to cancer diseases is increasing in the world. There is an urgent need to develop alternative therapeutic measures against the disease. Our study reports the cytotoxicity activity of Garcina epunctata (gutifferae) in human promyelocytic leukemia cells (HL-60) and prostate cancer cells (PC-3) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and morphological changes associated with apoptosis were examined by flow cytometry and Hoescht staining respectively. The results of in vitro antiproliferative screening of fractions and extract from G. epunctata indicated that three fractions inhibited the viability of PC-3 cells with IC₅₀ varied from 50 to 88 μ/ml while two fractions inhibited the proliferation of HL-60 cells with IC₅₀ range between 47.5 and 12 μg/ml. Among the entire fraction tested, Hex-EtOAc (75:25) showed cytotoxic effects on the two cell lines and EtOAc fraction was most active only HL-60 cells (12 μg/ml). Treatment of HL-60 cells with G. epunctata (20, 50, 100 μg/ml) for 24 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a number of apoptotic bodies containing nuclear fragments were observed in cells treated with 100 μg/ml. The EtOAc fraction of G. epunctata treatment significantly arrested HL-60 cells at the G0/G1 phase (p < 0.05) and ROS was significantly elevated as well as the loss of membrane mitochondrial potential in a concentration dependant manner. The results demonstrated that the EtOAc fraction of G. epunctata inhibited the proliferation of HL-60 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway.     

Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos

Prenatal exposure to alcohol promotes the level of reactive oxygen species within embryos and results in developmental disorders. In this study, we investigated the effect of capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient in red peppers, on ethanol-induced teratogenicity in mouse embryos (embryonic days 8.5–10.5). In response to ethanol administration (1.0 μl/ml), developmental parameters such as yolk sac circulation, allantois, heart, hindbrain, midbrain, forebrain, otic and optic systems, branchial bar, olfactory system, forelimb, hindlimb, and somites decreased significantly in comparison with those of control group (p < 0.05). However, the concurrent administration of capsaicin (1 × 10⁻⁸ μg/ml or 1 × 10⁻⁷ μg/ml) and ethanol significantly ameliorated most of the morphological scores excepting yolk sac circulation and hindlimb scores (p < 0.05). Furthermore, the levels of superoxide dismutase activity and cytoplasmic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNAs in the ethanol-treated embryos recovered to the levels observed in control embryos by capsaicin co-administration. These results indicate that capsaicin has a protective effect against ethanol-induced teratogenicity via an antioxidative activity.     

Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: Assessment of viability,sperm chromatin structure stability,ROS production and motility

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 × 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 × 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.     

In vitro assessment of the adverse effects of antiretroviral drugs on the human male gamete

This study was designed to investigate the in vitro effects of didanosine, zidovudine, saquinavir and indinavir, commonly used in highly active antiretroviral therapy, on human sperm fertility parameters. Thirty semen samples from healthy men were collected and prepared by gradient density method. Aliquots of 90% fractions with >80% motile spermatozoa were incubated for 1, 3, and 6 h with different concentrations of the antiretroviral drugs (20, 40, and 80 μg/ml). Sperm motility was evaluated by computer assisted sperm analysis system. Sperm mitochondrial potential was evaluated using 3,3′-dihexyloxacarbocyanine iodide (DIOC₆) and the acrosome reaction was examined using pisum sativum agglutinin method. A dose-dependent decrease in sperm motility was observed with saquinavir. Saquinavir also induced a significant time and dose-dependent decrease in mitochondrial potential and an increase in spontaneous acrosome reaction. These findings indicate that, in vitro, higher doses of saquinavir have adverse effects on sperm motility, mitochondrial potential and acrosome reaction.     

The effect of Phloretin on human γδ T cells killing colon cancer SW-1116 cells

ObjectiveTo explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells.Methodsγδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48 h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells.ResultsAfter cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31 ± 3.00% to 78.40 ± 10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35 μg/ml to 18.75 μg/ml, it could significantly proliferate the γδ T cell growth (P < 0.05) and inhibit the growth of SW-1116 cells in dose–response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P < 0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P < 0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group.ConclusionPhloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity.     

Protective effects of dehydrocostus lactone against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells

Oxidative stress regulates cellular functions in multiple pathological conditions, including bone formation by osteoblasic cells. To elucidate the protective effects of dehydrocostus lactone on the response of osteoblast to oxidative stress, osteoblastic MC3T3-E1 cells were incubated with 0.3 mM hydrogen peroxide (H₂O₂) and/or dehydrocostus lactone (0.1–10 μg/ml), and markers of osteoblast function and oxidative damage were examined. Dehydrocostus lactone (0.1–10 μg/ml) significantly increased osteoblast growth compared with control (P < 0.05). H₂O₂-induced reduction of differentiation markers such as alkaline phosphatase (ALP), collagen content, and calcium deposition was recovered in the presence of dehydrocostus lactone (0.4–2 μg/ml). Treatment with dehydrocostus lactone (10 μg/ml) decreased the production of osteoclast differentiation-inducing factors such as interleukin (IL)-6 and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of H₂O₂. Moreover, dehydrocostus lactone (0.4–2 μg/ml) decreased the formation of protein carbonyl (PCO) and malondialdehyde (MDA) induced by H₂O₂ in osteoblasts. Taken together, these results demonstrate that dehydrocostus lactone can protect osteoblasts against H₂O₂-induced cellular dysfunction. These results also suggest that dehydrocostus lactone may be valuable as a protective agent against oxidative damage in osteoblasts.     

A randomized,controlled trial of a clinical pharmacist intervention in microdiscectomy surgery – Low dose intravenous ketamine as an adjunct to standard therapy

AimThe hypothesis that postoperative pain would be reduced by using 1 μg/kg/min of ketamine, both intra- and post-operatively, for lumbar microdiscectomy surgery was assessed by measuring morphine consumption. Patient side effects were reported.MethodsForty-five patients undergoing microdiscectomy surgery were randomized under double-blind conditions into three groups: Group1 (G1) received normal saline, Group 2 (G2) ketamine (1 μg/kg/min) intra-operatively and Group 3 (G3) ketamine (1 μg/kg/min) both intra- and post-operatively. Morphine consumption, pain scores, nausea and vomiting, CNS disorders were recorded for 24 h post surgery. This study was conducted by applying the concept of a clinical pharmacist intervention.ResultsThe time for the first analgesia demand dose was significantly shorter (P < 0.05) in G117 ± 1.7 min than for G2 and G3. In G3 morphine consumption 6, 12, and 24 h after surgery was 3 ± 2.26, 9.2 ± 2.11 and 26.9 ± 2.71 mg. Total morphine consumption was significantly lower for G3 than for G1 or G2 (P < 0.05). The visual analog scale score (VAS) values were significantly lower in G3 (P < 0.05) than for the other groups during the first 24 h. The rate of nausea and vomiting was significantly higher in G1 vs G3 (P < 0.05). No difference in drug induced CNS disturbances was observed among the groups.ConclusionsUsing 1 μg/kg/min of ketamine hydrochloride intra- and post-operatively for microdiscectomy surgery could be an adjunct therapy to reduce postoperative morphine consumption minimizing its side effects.Collaborative clinical pharmacy practice on the basis of pharmacology had an effective role in improving the general outcome of microdiscectomy surgery.     

Effect of prenatal manganese intoxication on [3H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine

In the present study we examined the effects of prenatal manganese (Mn) intoxication on [³H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine (6-OHDA). MnCl₂ ? 4H2O (10,000 ppm) was added to the drinking water of pregnant Wistar rats for the duration of pregnancy. On the day of parturition, Mn was discontinued as an additive to the drinking water. The control group consisted of rats that consumed water without Mn. Three days after birth, rats in both groups (control and Mn) were pretreated with desipramine hydrochloride (20 mg/kg) and pargyline hydrochloride (50 mg/kg) and injected bilaterally icv with one of three doses of 6-OHDAhydrobromide (15 μg, 30 μg or 67 μg base form in saline on each side) or with saline (control). 6-[³H]-Dglucose (500 μCi/kg, ip) was administered to male offspring in adulthood; after 15 min, brain specimens were taken (frontal cortex, hippocampus, striatum, thalamus with hypothalamus, pons and cerebellum) for determination of radioactivity in a liquid scintillation counter. Low dose 6-OHDA (15 μg icv) increased [³H]glucose uptake in all brain regions (p < 0.05) in both control and Mnintoxicated animals. In rats lesioned with a moderate dose of 6-OHDA(30 μg icv), [3H]glucose uptake was unaltered in both control and Mn-exposed rats. High dose 6-OHDA(67 μg icv) reduced [³H]glucose uptake in all brain regions of Mn-exposed rats (except for cerebellum) compared with the saline group (all, p < 0.05). There was no change in regional brain uptake of [³H]glucose in control rats. In conclusion, this study shows that mild neuronal insult (15 μg icv 6-OHDA) increased glucose uptake in the brain while severe damage (concomitant 60 μg icv 6-OHDA and Mn treatment) significantly diminished this process.     

Physiological interactions between the hypothalamic-pituitary-gonadal axis and spleen in rams actively immunized against GnRH

Hypothalamic-pituitary-gonadal (HPG) axis is strongly implicated in the regulation of immune system. The objective was to determine the effects of immunocastration on splenic reproduction- and immunity-related gene expressions, and serum cytokine profiles in rams. Forty rams were randomly allocated into three groups: control (n = 14); surgically castrated (n = 13); or immunized (n = 13) against 100 μg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 6 months of age (with a booster 2 months later). Blood samples (for hormone and immune cytokine profiles) were collected at 1-month intervals until rams were slaughtered (10 months). Compared to intact controls, anti-GnRH immunization reduced (P < 0.05) serum concentrations of LH, FSH, and testosterone. Reduced testosterone abrogated its inhibitor feedback effect on the synthesis of GnRH in spleen, as evidenced by increased (P < 0.05) protein content and mRNA expressions of GnRH, and simultaneously decreased (P < 0.05) mRNA expressions of androgen receptor in spleen. In parallel with the increased GnRH production in spleen, the mRNA expressions of interleukin (IL)-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α) as well as lymphocyte marker CD4, CD8 and CD19 molecules were increased (P < 0.05) in spleen. Consistently, serum concentrations of IL-2, IL-4, IL-6, TNF-α were increased (P < 0.05) in rams following immunization. Similarly, deprivation of testosterone by surgical castration also increased (P < 0.05) GnRH and thus immune cytokine expressions in spleen. Collectively, our data suggested that immunocastration increased GnRH production in spleen by abrogating the inhibitory feedback effects from testosterone, consequently improving the immune markers of spleen and serum immune cytokines in rams.     

Does potassium sorbate induce genotoxic or mutagenic effects in lymphocytes?

The present study evaluates the genotoxic potential of potassium sorbate (PS) in cultured and isolated human lymphocytes. To assess the damage caused by PS in humans, we designed in vitro experiments by measuring chromosomal aberrations (CAs), sister-chromatid exchanges (SCEs), micronucleus (MN) and comet assays. Lymphocytes were treated with negative control (sterile distilled water), positive control (MMC for cultured lymphocytes, and H₂O₂ for isolated lymphocytes) and four concentrations (125, 250, 500, and 1000 μg/ml) of PS. According to the results, PS treatment significantly increases the CAs (with or without gaps at 500 and 1000 μg/ml concentrations) and SCEs (at 250, 500, 1000 μg/ml for 24 h and 125, 250, 500, 1000 μg/ml for 48 h) compared with vehicle control. Following treatment of the isolated lymphocytes for 1 h, significant PS-induced DNA strand breaks were observed, at all concentrations. However, PS failed to significantly affect the MN assay. On the contrary, PS does not cause cell cycle delay as noted by the non-significant decrease in the cytokinesis-block proliferation index (CBPI) and replicative index (RI). Only a slight decrease was observed in the mitotic index (MI) at the highest concentration for both treatment times. From the results, PS is clearly seen to be genotoxic to the human peripheral blood lymphocytes in vitro.     

The in vitro effect of duroquinone on functional competence,genomic integrity,and oxidative stress indices of sterlet (Acipenser ruthenus) spermatozoa

The sturgeon is a highly endangered fish species mostly due to over-fishing, habitat destruction, and water pollution. Duroquinone (derivative of 1,4-benzoquinone) is a xenobiotic compound widespread in the environment. The effect of duroquinone on motility, DNA integrity, and oxidative stress indices in sterlet, Acispenser ruthenus, spermatozoa was investigated. Sterlet sperm was exposed for 2 h to duroquinone at concentrations of 25, 50, 100, and 150 μM. Spermatozoa motility, velocity, and ATP content were significantly decreased with exposure to duroquinone. The level of DNA damage significantly increased at concentrations of 50 μM and above. Oxidative stress indices (lipid peroxidation and content of carbonyl proteins) and superoxide dismutase (SOD) activity increased significantly with increasing concentrations of duroquinone. Oxidative stress in sterlet spermatozoa induced by duroquinone was shown to impair spermatozoa DNA integrity, motility parameters, and the antioxidant defense system. Spermatozoa motility, content of carbonyl proteins, and SOD activity were shown to be sensitive biomarkers, exhibiting strong responses to low concentrations of the xenobiotic. Results also suggested that fish spermatozoa in vitro assays may provide a simple and efficient means of monitoring residual pollutants in the aquatic environment.