Laifu Zhong

Curcumin attenuates acrylamide-induced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging.

Acrylamide (AA), a proven rodent carcinogen, has recently been discovered in foods heated at high temperatures. This finding raises public health concerns. In our previous study, we found that AA caused DNA fragments and increase of reactive oxygen species (ROS) formation and induced genotoxicity and weak cytotoxicity in HepG2 cells. Presently, curcumin, a natural antioxidant compound present in turmeric was evaluated for its protective effects. The results showed that curcumin at the concentration of 2.5 microg/mL significantly reduced AA-induced ROS production, DNA fragments, micronuclei formation, and cytotoxicity in HepG2 cells. The effect of PEG-catalase on protecting against AA-induced cytotoxicity suggests that AA-induced cytotoxicity is directly dependent on hydrogen peroxide production. These data suggest that curcumin could attenuate the cytotoxicity and genotoxicity induced by AA in HepG2 cells. The protection is probably mediated by an antioxidant protective mechanism. Consumption of curcumin may be a plausible way to prevent AA-mediated genotoxicity.

Oxidative DNA Damage in Relation to Neurotoxicity in the Brain of Mice Exposed to Arsenic at Environmentally Relevant Levels

Oxidative DNA Damage in Relation to Neurotoxicity in the Brain of Mice Exposed to Arsenic at Environmentally Relevant Levels: Fengyuan Piao, et al. Department of Hygiene, Dalian Medical University, China—To clarify the association between oxidative DNA damage and the neurotoxicity of arsenic, the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) as an index of oxidative DNA damage in the brain was examined in mice fed with drinking water containing 1 or 2 ppm arsenic, using an HPLC‐electrochemical detector and immunohistochemical method. 8‐OHdG levels were significantly increased in the brain of mice given arsenic and its immunoreactivity was distributed in the cerebral and cerebellar cortexes. Cerebral cortex neurons and Purkinje cells in the cerebellar cortex showed degenerative changes in accordance with the distribution of 8‐OHdG immunoreactivity. The levels of arsenic in this study were lower than those reported in epidemiological studies. Thus, we conclude that environmentally relevant levels of arsenic induce pathological changes through oxidative DNA damage in the brain tissues in vivo and that cerebral and cerebellar cortex neurons seem to be the major targets of arsenic neurotoxicity.

The role of oxidative stress in deoxynivalenol-induced DNA damage in HepG2 cells.

Deoxynivalenol (DON) is a trichothecene mycotoxin and a cereals contamination, whose cytotoxicity has been shown in animals and various cells. However, with respect to the deoxynivalenol-induced DNA damage, especially in humans, are not well understood. The aim of this study was to assess the role of oxidative stress in deoxynivalenol-induced DNA damage, using human hepatoma HepG2 cells. Exposure of the cells to DON caused significant increase of DNA migration in comet assay at concentrations of 3.75-30 microM, which suggests that DON caused DNA strand breaks. To elucidate the role of antioxidation in those effects, DNA migration was monitored by pre-treatment with hydroxytyrosol (HT) as an antioxidant in comet assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. The DNA damage induced by DON was almost completely prevented. In order to clarify the underlying mechanisms, we evaluated the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. Significant increase in the level of ROS was observed in HepG2 cells at a higher concentration (60 microM). The involvement of lipid peroxidation in the DNA damage of DON was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), the doses being 7.5-60 microM and 3.75-15 microM, respectively. These results indicate that the DNA damage induced by DON in HepG2 cells is probably related to the oxidative stress.

Effects of hydroxytyrosol-20 on carrageenan-induced acute inflammation and hyperalgesia in rats.

Hydroxytyrosol (HT) is a simple phenol compound extracted from olive leaves. The content of HT in the studied preparation was about 20%, and the preparation was called hydroxytyrosol‐20 (HT‐20). HT has antioxidant and antiinflammatory activities. There has been no report so far on the efficacy of HT‐20 in carrageenan‐induced acute inflammation and hyperalgesia in rats. Therefore, the aim of this study was to assess the inhibitory role of HT‐20 on carrageenan‐induced swelling and hyperalgesia of rat paw. Paw inflammation was assessed by the increase in paw volume and hyperalgesia. The rat paws were cut out under ether anesthesia at 270 min after administration of carrageenan. The tissue of the right paw was isolated separately from the individual rat. The levels of the tumor necrosis factor‐alpha (TNF‐α), interleukin 1β (IL‐1β) and interleukin 10 (IL‐10) mRNA in the tissue were estimated by reverse transcription‐polymerase chain reaction (RT‐PCR). The results showed that the paw pressure thresholds of rats orally administered HT‐20 significantly increased at 210, 240 and 270 min after administration of carrageenan, compared with corresponding basal paw pressure thresholds; the degree of swelling of the right hind paw showed a statistically significant reduction, compared with rats in the carrageenan‐treated control. In this model, HT‐20 appears to decrease pro‐inflammatory cytokines IL‐1β and TNF‐α and not to increase the antiinflammatory cytokine mRNA expression of IL‐10. Copyright

Genotoxic effect of 6-gingerol on human hepatoma G2 cells

6-gingerol, a major component of ginger, has antioxidant, anti-apoptotic, and anti-inflammatory activities. However, some dietary phytochemicals possess pro-oxidant effects as well, and the risk of adverse effects is increased by raising the use of doses. The aim of this study was to assess the genotoxic effects of 6-gingerol and to clarify the mechanisms, using human hepatoma G2 (HepG2) cells. Exposure of the cells to 6-gingerol caused significant increase of DNA migration in comet assay, increase of micronuclei frequencies at high concentrations at 20-80 and 20-40 microM, respectively. These results indicate that 6-gingerol caused DNA strand breaks and chromosome damage. To further elucidate the underlying mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH). In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-hydroxydeoxyguanosine (8-OHdG). Results showed that lysosomal membrane stability was reduced after treatment by 6-gingerol (20-80 microM) for 40 min, mitochondrial membrane potential decreased after treatment for 50 min, GSH and ROS levels were significantly increased after treatment for 60 min. These suggest 6-gingerol induces genotoxicity probably by oxidative stress; lysosomal and mitochondrial damage were observed in 6-gingerol-induced toxicity.

Protective effect of hydroxytyrosol against acrylamide-induced cytotoxicity and DNA damage in HepG2 cells.

The chemoprotective effect of hydroxytyrosol (HT) against acrylamide (AA)-induced cytotoxicity and DNA damage was investigated in a human hepatoma cell line, HepG2. The cytotoxicity was estimated by methyl thiazol tetrazolium bromide (MTT) assay. The comet assay was used to monitor DNA damage. The intracellular reactive oxygen species (ROS) formation and the level of oxidative DNA damage were estimated by using 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe and by using immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG). Intracellular glutathione (GSH) level was estimated by fluorometric methods. The results showed that HT significantly reduced the cytotoxicity, DNA damage, intracellular ROS formation and 8-OHdG level caused by AA in a concentration-dependent manner. It was also found that HT concentration-dependently attenuated GSH depletion in HepG2 cells treated with 10mM AA. These findings suggest that HT has a strong protective ability against the cytotoxicity and DNA damage caused by AA.

Possible involvement of oxidative stress in potassium bromate-induced genotoxicity in human HepG2 cells

Potassium bromate (KBrO(3), PB) is a by-product of ozone used as disinfectant in drinking water. And PB is also a widely used food additive. However, there is little known about its adverse effects, in particular those related to its genotoxicity in humans. The aim of this study was to investigate the genotoxic effects of PB and the underlying mechanisms, using human hepatoma cell line, HepG2. Exposure of the cells to PB caused a significant increase of DNA migration in single cell gel electrophoresis (SCGE) assay and micronuclei (MN) frequencies in micronucleus test (MNT) at all tested concentrations (1.56-12.5 mM and 0.12-1 mM), which suggested that PB-mediated DNA strand breaks and chromosome damage. To indicate the role of antioxidant in those effects, DNA migration was monitored by pre-treatment with hydroxytyrosol (HT) as an antioxidant in SCGE assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. To elucidate the genotoxicity mechanisms, the study monitored the levels of reactive oxygen species (ROS), glutathione (GSH) and 8-hydroxydeoxyguanosine (8-OHdG). PB was shown to induce ROS production (12.5 mM), GSH depletion (1.56-12.5 mM) and 8-OHdG formation (6.25-12.5 mM) in HepG2 cells. Moreover, lysosomal membrane stability and mitochondrial membrane potential were further studied for the mechanisms of PB-induced genotoxicity. A significant increase was found in the range of 6.25-12.5 mM in lysosomal membrane stability assay. However, under these PB concentrations, we were not able to detect the changes of mitochondrial membrane potential. These results suggest that PB exerts oxidative stress and genotoxic effects in HepG2 cells, possibly through the mechanisms of lysosomal damage, an earlier event preceding the oxidative DNA damage.

The Protective Effects of Hydroxytyrosol Against UVB-induced DNA Damage in HaCaT cells

The chemoprotective effect of hydroxytyrosol (HT) against UVB‐induced DNA damage was investigated in a human skin keratinocyte cell line, HaCaT. The comet assay was used to monitor DNA strand breaks. Intracellular reactive oxygen species (ROS) formation was measured by flow cytometry using 2,7‐dichlorofluorescein diacetate (DCFH‐DA). The levels of oxidatively generated damage to DNA were estimated by immunocytochemistry analysis of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG). The protein expression of p53 and NF‐κB was estimated by western blotting. The results showed that HT significantly reduced the DNA strand breaks caused by UVB. It was also found that HT reduced intracellular ROS formation and 8‐OHdG level caused by UVB. Furthermore, HT attenuated the expression of p53 and NF‐κB in a concentration‐dependent manner. These results strongly suggest that HT has a significant protective ability against UVB‐induced DNA damage and that oxidative stress plays an important part in it. Copyright

Possible involvement of oxidative stress in trichloroethylene-induced genotoxicity in human HepG2 cells

Trichloroethylene (TCE) is an environmental and industrial pollutant whose hepatotoxicity has been demonstrated in experimental animals. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of TCE and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. Exposure of the cells to TCE caused significant increase of DNA migration in comet assay and of micronuclei (MN) frequencies at all tested concentrations (0.5-4mM), respectively, which suggests that TCE caused DNA strand breaks and chromosome damage. The involvement of lipid peroxidation in the genotoxic properties of TCE was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). To elucidate the role of glutathione (GSH) in these effects, the intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by co-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to TCE-induced cytotoxicity and DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by TCE was almost completely prevented. These results indicate that TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.

Boric acid inhibits LPS-induced TNF-α formation through a thiol-dependent mechanism in THP-1 cells

Oxidative stress plays an important role during inflammatory diseases and antioxidant administration to diminish oxidative stress may arrest inflammatory processes. Boron has been implicated to modulate certain inflammatory mediators and regulate inflammatory processes. Here we investigated the role of the tripeptide glutathione (GSH) in modulating the effects of boric acid (BA) on lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) formation in THP-1 monocytes. Interestingly, we found that BA had no significant effects on both TNF-alpha production and intracellular GSH contents, whereas it could inhibit LPS-induced TNF-alpha formation and ameliorated the d,l-buthionine-S,R-sulfoximine (BSO)-induced GSH depletion. Twenty-four hour incubation with BSO induced a decrease of the intracellular GSH and an increase of TNF-alpha. Treatment with N-acetyl-l-cysteine (NAC) did not significantly increase intracellular content of GSH but significantly reduced the secretion of TNF-alpha. BSO-pretreatment for 24h enhanced the LPS-induced secretion and mRNA expression of TNF-alpha further. BA inhibited LPS-stimulated TNF-alpha formation was also seen after GSH depletion by BSO. These results indicate that BA may have anti-inflammatory effect in the LPS-stimulated inflammation and the effect of BA on TNF-alpha secretion may be induced via a thiol-dependent mechanism.