Cadmium Induced Cellular Adaptive Response And Its Molecular Mechanism

The adaptive response (AR) is a phenomenon where cells pretreated with a sub-genotoxic low dose of mutagen become significantly resistant to a subsequent higher dose of genotoxic agent. AR has been studied mainly with low doses of radiation and several notable findings have been found which may help to understand the mechanism. However, more information is needed for cross adaptation. Human health is threatened not only by radiation but also by environmental chemical toxicants, and cross adaptation and synergistic sensitization may play important roles in modifying the biological effect caused by environmental factors to which we are exposed in our daily life, thus the health risk caused by environmental factors should be assessed in the context of combined exposure Cadmium is one of the most frequent environmental toxins. The wide use in industry and persistence in environment make cadmium one of the hot spots in the toxicology research field. Thus, to investigate the AR, especially the cadmium induced AR will help us to deeper understand the molecular mechanisms of health risk effects caused by combined exposure to environmental physical and chemical factors and to provide theoretical support to correctly assess the health risk effects and restore the model of the cancer risk assessment.Using three Chinese hamster ovary (CHO) cell lines with different repair capacity of DNA damage, different doses of cadmium and y-ray radiation as the priming and challenging treatments and micronuclei formation as the biological endpoint, we observed the cadmium induced adaptive/ cross adaptive response and the magnitude of AR in these cell lines to elucidate the influence of DNA repair capacity, the priming and challenging doses on the cadmium induced AR. Meanwhile, we also explored the roles of ataxia-telangiectasia mutated (ATM) and H2S in this response. Furthermore, we used human liver cell lines to investigate the effect of NaHS, the donor of exogenous H2S, and the inhibitors of endogeous H2S production on this response. Moreover, the cellular ROS level was meausured under different treatments as well as the expression level of CSE and CBS, to elucidate the role of H2S and its two key enzymes for endogenous synthesization of H2S in cadmium induced AR. Finally, using westrn blot and real-time PCR, we observed the interaction among H2S, ATM, P53 and NF-?B to explore the the molecular mechanism of H2S mediateing AR induced by cadmium. In the experiment conducted in CHO cell lines, our data showed that pre-exposure to cadmium at non-lethal concentration could induce AR or cross AR in all the cell lines toward subsequent challenge of cadmium or irradiation. When challenged by 50?M CdCl2, compared with the cells treated with high dose of CdCl2 alone, the yield of micronucle (YMN) decreased by 52%,40% and 52% in CHO-9, EM-C11 and XR-C1 cell lines, respectively, indicating CHO-9 and its two mutant cell lines exhibited significant AR, but the magnitude of this response in EM-C11 was less evident than that in the other two cell lines. When the challenge dose increased to 100?M, cadmium induced AR was only observed in CHO-9 cells, while the other two cell lines, EM-C 11 and XR-C1, just showed slight and insignificant AR. As to cadmium induced cross AR, when cells were challenged by 1Gy y-radiation, all of these cell lines exhibited AR whether they were primed with 0.1 or 1?M of CdCl2. Interestingly, with respect to 2Gy of irradiation, when the cells were pretreated with 0.1?M of cadmium, AR was only observed in CHO-9; when the priming dose of cadmium increased to 1?M, AR was observed in both CHO-9 and EM-C 11 but with different magnitude,where the YMN were decreased by 22% in CHO-9and 12% in EM-C11, respectively, compared with the cells treated with 2Gy radiation alone, However, this response was almost absent in the irradiated XR-C1 cells pretreated with either 0.1 or 1?M of cadmium. In addition, treatment of cells with PI-3K inhibitor wortmannin eliminated the AR in all the cell lines and the same phenomena were also observed when cells were treated with ATM specific inhibitor KU-55933 or endogenous H2S synthesis inhibitor PPG during the priming treatment and the time interval. Moreover, H2S directly induced AR toward high dose of radiation in CHO-9 cell line.In the experiment conducted in human liver cell lines, our data showed that the cadmium ions with a sub-lethal concentration could also induce AR toward high dose of radiation in human liver cells. Under the same priming and challenging treatment, low dose of cadmium pre-treatment protected Chang liver cells against the damage caused by the subsequent high dose of radiation (p<0.01), while such protecting effect could not be observed in HepaG2 cells a. In addition, ATM kinase also participated the cadmium induced cross AR in Chang liver cells, for this response could be abrogated totally by the ATM inhibitor KU-55933.Recently, it has been demonstrated that H2S serves as a novel third gastransmitter in the body alongside nitrogen monoxide (NO) and carbon monoxide (CO). Our results showed that H2S might be also involved in cadmium induced AR. The pretreatment of Chang cells with 50?M NaHS decreased the YMN in Chang cell line by 32.65%, compared with the cells treated 2Gy radiation alone, indicating that H2S can directly stimulate a significant AR to radiation, and this response exhibited a priming-dose-dependent profile, for the magnitude of AR induced by 75?M NaHS was even more evident than that induced by 50?M NaHS, where the YMN wasdecreased by 44.85%. In mammalian cells, two pyridoxal-5'-phosphate-dependent enzymes, cystathionine?-lyase (CSE) and cystathionine?-synthase (CBS) are responsible for the majority of the endogenous H2S production. In liver cells, the expression of both CSE and CBS can be detected. We found that cadmium induced AR could be eliminated by the addition of DL-propargylglycine (PPG), the endogenous H2S synthetase inhibitor of CSE, but not by aminooxyacetic acid (AOAA), the inhibitor of CBS.The cellular ROS level was increased significantly by the treatment of 50?M H2S,75 or 100?M H2S exposure also increased the ROS level but insignificanly. When the cells were irradiation with 2Gy?-rays, the cellular ROS level increased by 2.5 times, compared to the control. When the cells were pretreated with H2S for 4h before exposed to 2Gy radiation, the ROS level was also ascended by 60%,74% and 75%, respectively, compared to the control, but was lower than that in the cells received radiation treatment alone, indicating that the pre-exposure of cells to H2S can inhibit the induction of ROS by radiation.Both cadmium treatment and irradiation up-regulated the expression of CSE protein in a time-dependent manner, which was increased by 81% and 134% at 12h after the cadmium or radiation treatment, while the expression of CBS protein remained at the control level after these treatments. In the primed cells, the time course of CBS expression showed no significant difference with the cells treated with 2Gy irradiation alone, however, the CSE expression was increased by 106% at 8h after radiation compared with the cells treated with radiation alone. The above results indicated that CSE, not CBS, was involved in the cadmium induced AR.Further, our data showed that high dose of irradiation could activate and up-regulate the phosphorylation of ATM and P53. The NF-?B activity was also up-regulated significantly by high dose of irradiation. In the process of the induction of cellular AR, there might be some interactions among H2S and the signaling factors mentioned above. In the cells challenged with 2Gy radiation, the protein level of p-ATM was decreased by the pre-treatment with exogenous H2S but increased by the pre-treatment with PPG immediately after radiation. The p-ATM expression was then comparative to the cells without any pre-treatment at other indicated time points. However, when the cells were treated with ATM kinase inhibitor before irradiation, the expression level of CSE mRNA and protein did not change significantly at various time points compared to the irradiated cells without inhibitor treatment. Besides, our data also showed that, compared to the cells treated with 2Gy irradiation alone, the phosphorylation level of P53 declined by 50% in the cells pre-treated with exogenous H2S, (p<0.01), while PPG treatment did not change the p-P53 expression level significantly. In addition, H2S may negatively regulate the activation of NF-?B by radiation. Our data showed that H2S treatment alone decreased the activity of NF-?B, while PPG treatment alone did not influence the activity of NF-?B. Compared with the cells treated with high dose of radiation alone, the activity of NF-?B was significantly down-regulated by pre-treatment of H2S before irradiation. However, the PPG pretreatment promoted the activation of NF-?B by radiation.In conclusion, the present results indicated that low dose of cadmium could induce AR or cross AR toward the same high dose of toxicant or high dose of radiation. The triggering of cadmium-induced AR was independent of DNA repair capacity while The magnitude of adaptive response (MAR) in adapted cells was related to multiple factors including DNA repair capacity, priming dose of cadmium and the challenging dose of cadmium or irradiation. Single strand break (SSB) or base excision repair (BER) rather than double strand break (DSB) repair was mainly involved in the AR. This cellular response toward further challenge of cadmium or irradiation may be mediated through ATM pathway.Our data also demonstrate firstly that H2S is involved in the cadmium induced cross-adaptive response to challenging radiation. CSE, rather than CBS, may mainly responsible for the H2S production during this response. However, the activation of CSE is independent of ATM but could negatively regulate the phosphorylation of ATM and P53. In addition, the activation of NF-?B by radiation could also be negatively regulated by H2S.