Ping-Chi Hsu

Antioxidant nutrients and lead toxicity

Lead-induced oxidative stress contributes to the pathogenesis of lead poisoning for disrupting the delicate prooxidant/antioxidant balance that exists within mammalian cells. Production of reactive oxygen species (ROS) is increased after lead treatment in in vitro studies. In vivo studies suggest that lead exposure causes generation of ROS and alteration of antioxidant defense systems in animals and occupationally exposed workers. The mechanisms for lead-induced oxidative stress include the effect of lead on membrane, DNA, and antioxidant defense systems of cells. From low to high doses of lead exposure, there are different responses of lead-induced oxidative stress in various target sites including lung, blood vessels, testes, sperm, liver, and brain in epidemiological as well as animal studies. Therefore, reducing the possibility of lead interacting with critical biomolecules and inducing oxidative damage, or bolstering the cells antioxidant defenses might be associated with the beneficial role of antioxidant nutrients through exogenous supplementation of antioxidant molecules. Although many researchers have investigated the benefit of antioxidants in preventing lead toxicity, the mechanisms of antioxidant nutrients being effective via rebalancing the impaired prooxidant/antioxidant ratio are not completely clear. Antioxidant nutrients including, vitamin E, vitamin C, vitamin B(6), beta-carotene, zinc, and selenium, are addressed in this review to discuss their beneficial role in lead-induced oxidative stress.

Semen quality after prenatal exposure to polychlorinated biphenyls and dibenzofurans

Large-scale poisoning occurred in central Taiwan in 1979 from ingestion of cooking oil contaminated by polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans. To determine whether in-utero exposure to these chemicals alters reproductive function, all prenatally exposed boys and appropriate controls were contacted for medical examination in 1998. Sperm of exposed children have increased abnormal morphology, reduced motility, and reduced capacity to penetrate hamster oocytes. Whether this will cause reduced fecundity, and how these effects can be extrapolated to the general population exposed to background levels of PCBs and dioxin-like chemicals, warrants further investigation.

Effects of vitamin E and/or C on reactive oxygen species-related lead toxicity in the rat sperm

This study was undertaken to investigate whether treatment with vitamin E (VE) and/or vitamin C (VC) protects rat sperm by inhibiting reactive oxygen species generation induced by lead (Pb) exposure. Male Sprague-Dawley rats were assigned to the following five groups: vitamin-unsupplemented; 150 mg VE/kg chow supplemented; 300 mg VE/kg chow supplemented; 500 mg VC/l drinking water supplemented and 150 mg VE/kg chow + 500 mg VC/l drinking water supplemented group. Rats in each group were divided into Pb-unexposed and Pb-exposed subgroups, received weekly intraperitoneal injection of 10 mg sodium acetate or 10 mg Pb acetate/kg for 6 weeks, respectively. The blood and sperm Pb levels were analyzed by graphite furnace atomic absorption spectrophotometer. Chemiluminescence was measured to evaluate the generation of sperm reactive oxygen species (ROS). Motility and sperm-oocyte penetration rate (SOPR) were measured. In Pb-unexposed rats, epididymal sperm counts, motility, ROS, and SOPR were not different in the five supplemented groups. Lead exposure might decrease the defense capacity of sperm to the oxidative stress and therefore elevate the ROS generation, reduce sperm motility, and reduce SOPR. Supplementation with VE and/or VC reduced ROS generation, prevented loss of motility and capacity of oocyte penetration in Pb-exposed rats. This study suggests that supplementation with VE and/or VC inhibits Pb-related ROS generation, protects spermatozoa from loss of motility and oocyte penetration capability.

6‐Shogaol suppressed lipopolysaccharide‐induced up‐expression of iNOS and COX‐2 in murine macrophages

Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, antinausea, anti-inflammatory, and anticarcinogenic properties. In this study, we investigated the inhibitory effects of 6-shogaol and a related compound, 6-gingerol, on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS. Western blotting and reverse transcription-PCR analyses demonstrated that 6-shogaol significantly blocked protein and mRNA expression of inducible NOS (iNOS) and COX-2 in LPS-induced macrophages. The in vivo anti-inflammatory activity was evaluated by a topical 12-O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin. When applied topically onto the shaven backs of mice prior to TPA, 6-shogaol markedly inhibited the expression of iNOS and COX-2 proteins. Treatment with 6-shogaol resulted in the reduction of LPS-induced nuclear translocation of nuclear factor-kappaB (NF kappaB) subunit and the dependent transcriptional activity of NF kappaB by blocking phosphorylation of inhibitor kappaB (I kappaB)alpha and p65 and subsequent degradation of I kappaB alpha. Transient transfection experiments using NF kappaB reporter constructs indicated that 6-shogaol inhibits the transcriptional activity of NF kappaB in LPS-stimulated mouse macrophages. We found that 6-shogaol also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2, but not p38 mitogen-activated protein kinase (MAPK). Taken together, these results show that 6-shogaol downregulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NF kappaB by interfering with the activation PI3K/Akt/I kappaB kinases IKK and MAPK.

Lead exposure causes generation of reactive oxygen species and functional impairment in rat sperm

The relationships between blood lead, sperm lead, sperm reactive oxygen species (ROS) level, and sperm fertile capability were investigated to understand the effects of lead exposure on sperm function and the mechanism of these effects. Male Sprague-Dawley rats, 7 weeks old, were randomly divided into control group and lead-treated group. The controls and lead-treated animals received intraperitoneal injection of 10 mg sodium acetate and 10 mg lead acetate/kg body weight, respectively, weekly for 6 or 9 weeks. The blood lead and epididymal sperm lead were analyzed by graphite furnace atomic absorption spectrophotometer. Chemiluminescence was measured to evaluate the generation of sperm ROS. Sperm-oocyte penetration rate (SOPR) was measured to evaluate sperm function. After 6 weeks of lead exposure, the rats had average blood lead levels of 32 microg/dl, sperm lead levels of 0.67 +/- 0.11 microg/10(9) sperm, unchanged epididymal sperm counts, percent of motile sperms, and motile epididymal sperm counts compared with control animals. However, after 9 weeks of lead exposure, the rats had average blood lead levels of 48.0 +/- 4.3 microg/dl, sperm lead levels of 0.88 +/- 0.16 microg/10(9) sperm, statistically lower epididymal sperm counts, and lower motile epididymal sperm counts. There was a good correlation between the blood lead and sperm lead(r2 = 0.946, P < 0.001). The sperms of lead-exposed rats produced significantly higher counts ofchemiluminescence than did those from the control rats (P < 0.001). The chemiluminescence counts were positively associated with sperm lead level (r2 = 0.613, P < 0.001). Epididymal sperm counts, motility and motile epididymal sperm counts were negatively associated with sperm chemiluminescence (r2 = 0.255, 0.152, and 0.299; P < 0.01, 0.05, and 0.01, respectively). The SOPR were positively associated with epididymal sperm counts, motility and motile epididymal sperm counts (r2 = 0.136, 0.285, and 0.264; P < 0.05, 0.01, and 0.001, respectively). The sperm chemiluminescence was negatively associated with SOPR (r2 = 0.519, P < 0.001). It is concluded that lead exposure probably affected the sperm function by activating one of the pathways of ROS generation.

LEAD-INDUCED CHANGES IN SPERMATOZOA FUNCTION AND METABOLISM

The relationships between sperm reactive oxygen species (ROS) generation, the capacitation process and acrosome reaction, and the spermoocyte penetration rate (SOPR) were investigated to understand the effect of lead toxicity on sperm functions and the mechanisms of these effects. Male Sprague-Dawley rats received weekly intraperitoneal injections of 20 mg or 50 mg lead acetate/kg or 20 mg or 50 mg sodium acetate/kg (control) for 6 wk. Serum testosterone was measured by radioimmunoassay. In cauda epididymal spermatozoa, the chemiluminescence was measured to evaluate the sperm ROS generation. Chlortetracycline fluorescence assay was used to study the status of capacitation and acrosome reaction on fresh cauda epididymal spermatozoa and after 2, 4, or 24 h of incubation with 5 mg/ml bovine serum albumin. In lead-exposed rats, the serum testosterone levels were reduced, and the percentage of capacitation and the chemiluminescence were significantly increased in fresh cauda epididymal spermatozoa. The serum testosterone levels were negatively associated with the percentage of acrosome-reacted spermatozoa. Sperm chemiluminescence was positively correlated with the percentage of both capacitated and acrosome-reacted spermatozoa. The SOPR was negatively associated with the percentage of both capacitated and acrosome-reacted spermatozoa. In summary, this study showed that male rats exposed to lead had decreased serum testosterone levels and that this metal produced early onset of capacitation by one of the pathways of ROS generation. These effects might consequently result in premature acrosome reaction and reduced zona-intact oocyte-penetrating capability.

The association between semen quality in workers and the concentration of di(2-ethylhexyl) phthalate in polyvinyl chloride pellet plant air

OBJECTIVE To investigate potential associations between semen quality in workers and the concentrations of di(2-ethylhexyl) phthalate (DEHP) in personal air collected from polyvinyl chloride (PVC) plants. DESIGN Cross-sectional study. SETTING PVC plants in Taiwan. PATIENT(S) Forty-five male workers employed in two PVC pellet plants. INTERVENTION(S) None. MAIN OUTCOME MEASUREMENT(S) Sperm concentration, motility, morphology, and chromatin DNA integrity were accessed. RESULT(S) The workers were divided into low- and high-DEHP-exposed groups in accordance with the median levels of DEHP (23.7 μg/m(3)) in personal air. In the high-DEHP-exposed group, significant increases were found in the tendency for sperm DNA denaturation (αT) induction, the DNA fragmentation index (DFI), and propensity for coffee drinking. After adjusting for coffee drinking, cigarette smoking, and age, personal air concentrations of DEHP showed positive associations with αT (β = 0.038) and DFI (β = 0.140) and negative associations with sperm motility (β = -0.227). CONCLUSION(S) This is the first study to demonstrate a link between DEHP concentration in ambient air and the adverse effects in sperm motility and chromatin DNA integrity. Given the current wide use of these PVC products, the implications for phthalates toxicity and occupational health could be considerable.

Hydrogen peroxide induces premature acrosome reaction in rat sperm and reduces their penetration of the zona pellucida

Recent studies have demonstrated that mammalian sperm are capable of generating reactive oxygen species (ROS) and that this activity is significantly accelerated in subfertile subjects. The observed decrease in penetration of zona-intact oocyte might be explained by chemical-induced ROS-related early onset of capacitation and premature acrosome reaction, but the mechanism is not clear. We determine whether zona-intact oocyte penetration capability in rat epididymal sperm was affected by premature acrosome reaction in rat sperm treated with hydrogen peroxide (H2O2) and calcium ionophore A23187 or H2O2 and lysophosphatidyl choline. Chlortetracycline fluorescence assay was used to study the status of acrosome reaction on epididymal sperm. The sperm-oocyte binding and penetration assay was used to evaluate the capability for zona pellucida penetration. There was a positive linear correlation between the frequency of acrosome-reacted sperm and capability of sperm-oocyte binding and penetration in zona-free oocytes. In the zona-intact oocytes, the sperm-oocyte penetration rate was suppressed as the proportions of acrosome-reacted sperm increased. In summary, this study showed that premature acrosome reaction reduced rat sperms capability of penetrating zona-intact oocytes. However, this reduction is not seen in zona-free oocytes. These findings may provide a basis for understanding the effects of sperm ROS generation on zona pellucida penetration in male reproductive toxicology.

Effect of smoking on blood lead levels in workers and role of reactive oxygen species in lead-induced sperm chromatin DNA damage.

OBJECTIVE To investigate whether cigarette smoking affects the blood lead levels (BLL) and whether exposure to lead introduces sperm chromatin DNA damage in factory workers. DESIGN Cross-sectional study. SETTING A battery plant in Taiwan. PATIENT(S) Eighty male workers employed within a battery plant. INTERVENTION(S) Standard semen analysis was performed according to the World Health Organization guidelines. MAIN OUTCOME MEASURE(S) Assessment of BLL, sperm chromatin DNA structure, reactive oxygen species generation and other conventional parameters of semen quality. RESULT(S) As compared with nonsmoking workers, the BLL were found to be considerably higher among smokers. Statistically significant differences were found in the sperm DNA denaturation (alphaT) induction and the percentage of sperm with increased DNA denaturation (COMP alphaT) in workers with moderate BLL (>or=25 microg/dL). After adjustment for smoking propensity, a positive correlation was discernible between BLL and alphaT, COMP alphaT, and morphologic abnormality. Furthermore, alphaT and COMP alphaT were also found to have positive correlations with sperm superoxide anion production. CONCLUSION(S) Workers with higher BLL were found to be at a higher risk of sperm morphologic abnormality and chromatin DNA integrity. These data are significant because they can facilitate the estimation of lead exposure in reproductive toxicology.

Developmental exposure to decabrominated diphenyl ether (BDE‐209): Effects on sperm oxidative stress and chromatin dna damage in mouse offspring

Polybrominated diphenyl ethers (PBDEs) are used as brominated flame retardants and have been found in human milk in recent years. This study investigates whether prenatal exposure to decabrominated diphenyl ether (BDE‐209) induces sperm dysfunction in male offspring. Pregnant CD‐1 mice were gavaged once daily with corn oil (control), 10, 500, and 1500 mg kg−1 body weight of BDE‐209 from day 0 of gestation to day 17. The outcomes of male reproductive parameters were assessed on postnatal day 71. Anogenital distance, sperm‐head abnormalities, and testicular histopathology were significantly affected in male offspring prenatally exposed to 1500 mg kg−1. Significant increases in the tendency for sperm DNA denaturation (αT) induction and the DNA fragmentation index (DFI) were found in those exposed to 10, 500, and 1500 mg kg−1 (P < 0.05). We observed a significant increase of sperm hydrogen peroxide (H2O2) generation in the 10 and 1500 mg/kg/day groups compared to the control group (P < 0.05). Although our findings suggested that the mechanisms underlying BDE‐209‐induced sperm DNA damage and H2O2 generation might not be represented as a dose‐response relationship, we found that the greater the excess production of sperm H2O2, the greater the sperm αT (r = 0.65, P = 0.0155) and DFI (r = 0.53, P = 0.002). In conclusion, developmental exposure to BDE‐209 induced sperm‐head abnormality, oxidative stress, chromatin DNA damage, and testicular histopathological changes. These findings suggest that BDE‐209‐induced male reproductive effects might involve the formation of sperm H2O2 which attacks nucleic acids via H2O2 generation.