Zongping Liu

Lead poisoning combined with cadmium in sheep and horses in the vicinity of non-ferrous metal smelters.

The diagnosis of lead poisoning combined with cadmium in sheep and horses living on farmland in the vicinity of non-ferrous metal smelters in Baiyin of Gansu province in China was based on laboratory findings as well as clinical signs. The concentrations of lead, cadmium, copper and zinc in soils, water, forages, feed and blood, hair and tissues of affected sheep and horses were determined in the surroundings of the smelters in Baiyin and controls. Haematological values were also measured. The concentrations of these elements in soils, forages, water and feed were significantly higher than those in the control area (P<0.01). The contents of lead and cadmium were 9.3 and 690 times in forages, respectively, 10 and 35.6 times in feed. The concentrations of lead and cadmium in blood, hair and tissues of the affected sheep and horses were significantly higher than reference values and control animals (P<0.01). The occurrence of anaemia in affected animals belonged to a hypochromic and microcytic pattern. Lead and cadmium intake levels, estimated according to the ingestion rates of forages, illustrates that the apported lead and cadmium through the ingestion of vegetation growing in the closest sites to the smelters were approximately 6.0 mg Pb/kg body wt./day and 1.1 mg Cd/kg body wt./day in horses, 21.4 mg Pb/kg body wt./day and 4.0 mg Cd/kg body wt./day in sheep, surpassing the fatal dosages for horses of 1.7 mg Pb/kg body wt./day and for sheep of 4.4 mg Pb/kg body wt./day and 1.0 mg Cd/kg body wt./day. It is, therefore, suggested that the disease of sheep and horses in this region is caused by lead poisoning combined with cadmium, mainly due to environment heavy metal pollution by industrial activity. Meanwhile, as such levels of contamination in animal food and grain pose a significant potential risk to human health, these results have formed the basis for subsequent research on levels of metal contamination in human tissues from affected populations. Thus, it can be seen that knowledge of lead and cadmium concentrations in livestock in this region is important for assessing the effects of pollutants on domestic animals themselves and contaminant intakes by humans.

Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca2+]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca2+]i and inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities. This Cd-induced elevation of [Ca2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca2+ homeostasis, by compromising ATPases activities and ER-regulated Ca2+, and this elevation in Ca2+ triggers the activation of the Ca2+-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in Leydig cells. This study investigated the effects of ZEA on rat Leydig cells. Results showed that ZEA at different doses significantly inhibited the growth of Leydig cells by inducing apoptosis. ZEA treatment upregulated Bax expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, followed by the cleavage of poly(ADP-ribose) polymerase (PARP), resulting in Leydig cell apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast to other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated Leydig cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity.

Mitochondrial permeability transition and its regulatory components are implicated in apoptosis of primary cultures of rat proximal tubular cells exposed to lead

Previous studies have already demonstrated that mitochondria play a key role in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells. To further clarify the underlying mechanism of Pb-induced mitochondrial apoptosis, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and its regulatory components in Pb-induced apoptosis in rPT cells. Mitochondrial permeability transition pore (MPTP) opening together with disruption of mitochondrial ultrastructure, translocation of cytochrome c from mitochondria to cytoplasm and subsequent caspase-3 activation were observed in this study, suggesting that MPT is involved in Pb-induced apoptosis in rPT cells. Simultaneously, Pb-induced caspase-3 activation and apoptosis can be significantly inhibited by three MPTP inhibitors (CsA, DIDS, BA), which target different regulatory components of MPTP (Cyp-D, VDAC, ANT), respectively, demonstrating that Cyp-D, VDAC and ANT participate in MPTP regulation during lead exposure. Moreover, decreased ATP levels and increased ADP/ATP ratio induced by lead treatment can be significantly reversed by BA, indicating that Pb-mediated ANT dysfunction resulted in ATP depletion. In addition, up-regulation of VDAC-1, ANT-1 together with down-regulation of Cyp-D, VDAC-2 and ANT-2 at both the levels of transcription and translation were revealed in rPT cells under lead exposure conditions. In conclusion, Pb-mediated mitochondrial apoptosis in rPT cells is dependent on MPTP opening. Different expression levels in each isoform of three regulatory components contribute to alteration in their functions, which may promote the MPTP opening.

Induction of cytoprotective autophagy in PC-12 cells by cadmium.

Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.

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.

Calcium–calmodulin signaling elicits mitochondrial dysfunction and the release of cytochrome c during cadmium-induced apoptosis in primary osteoblasts

Cadmium (Cd) is a toxic heavy metal used in industry and is associated with adverse effects on human health following long- or short-term environmental exposure. Although Cd is known to induce apoptosis in many human organ systems, the mechanism that underlies its toxicity in primary osteoblasts (OBs) is not yet established. In the present study, we confirmed that Cd induced apoptosis in OBs isolated from the craniums of fetal Sprague-Dawley rats. We then showed that exposure to Cd transiently increased intracellular calcium ([Ca(2+)]i) levels for up to 1.5h, after which the levels returned to normal. Pretreatment with the calcium chelator BAPTA-AM was able to prevent Cd-induced apoptosis by reversing Cd-induced changes in the mitochondrial transmembrane potential (ΔΨm). In addition, we found that the antagonist of calcium-dependent calmodulin (CaM), W-7, inhibited the conformational change of calmodulin induced by Cd. Furthermore, Cd-induced apoptosis could be inhibited by W-7 through the suppression of the mitochondrial release of cytochrome c to the cytosol and the reversal of Cd-activation of caspase-3. These data indicate that activated Ca(2+)/CaM might transmit apoptotic signals to the mitochondria during Cd-induced apoptosis. Our findings provide new insights into the mechanisms underlying apoptosis in OBs following exposure to Cd.

Antioxidative effects of hesperetin against lead acetate-induced oxidative stress in rats

Objectives: Oxidative stress with subsequent lipid peroxidation (LP) has been suggested as a mechanism for lead-induced toxicity. The current study was carried out to evaluate antioxidant activity of hesperetin against lead acetate-induced oxidative stress. Materials and Methods: The male rats were treated with hesperetin in combination with lead acetate (500 mg/L). Results: The results indicated that hesperetin alone did not induce any significant changes in the biochemistry of serum, liver, and kidney tissues. On the other hand, lead-induced oxidative stress as indicated by significant changes in serum biochemical parameters, including increased lipid peroxide and decreased reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels in liver and kidney tissues. Hesperetin succeeded in improving these biochemical parameters towards the normal values of control. Conclusions: It suggests that hesperetin shows antioxidant activity and plays a protective role against lead-induced oxidative damage in liver and kidney of rats.

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway.

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.

Protective effect of N-acetylcysteine on experimental chronic cadmium nephrotoxicity in immature female rats

In the present study, female Sprague-Dawley rats received CdCl2 (50 mg/L through drinking water) and/or N-acetylcysteine (NAC, 120 mg/kg/day, orally) to investigate the protective effect of NAC on Cd-induced renal damage. Renal toxicity was evaluated by measuring the contents of total protein, β2-microglobulin, and α1-microglobulin in the urine and urinary enzyme markers of tubular necrosis, as well as levels of serum urea nitrogen and serum creatinine. Activities of antioxidant enzymes and contents of glutathione, malondialdehyde, and trace elements in the kidney were also measured. Animals that received both Cd and NAC showed a better renal function than those receiving Cd alone. In addition, NAC significantly reduced the levels of lipid peroxidation (LPO) in the kidney of cadmium-treated rats. The enzymic and nonenzymatic antioxidants levels are not restored, but their further decrease is prevented by NAC. Also NAC administration does not modify the urinary excretion of cadmium or contents of cadmium in the serum and kidney. In conclusion, NAC exerts its protective effect by decreased LPO and improving antioxidants status to prevent renal tubular damage induced by chronic Cd administration, most probably through its antioxidant properties.