Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE-cadherin.

The use of nanomaterials has raised safety concerns, as their small size facilitates accumulation in and interaction with biological tissues. Here we show that exposure of endothelial cells to TiO2 nanomaterials causes endothelial cell leakiness. This effect is caused by the physical interaction between TiO2 nanomaterials and endothelial cells' adherens junction protein VE-cadherin. As a result, VE-cadherin is phosphorylated at intracellular residues (Y658 and Y731), and the interaction between VE-cadherin and p120 as well as ß-catenin is lost. The resulting signalling cascade promotes actin remodelling, as well as internalization and degradation of VE-cadherin. We show that injections of TiO2 nanomaterials cause leakiness of subcutaneous blood vessels in mice and, in a melanoma-lung metastasis mouse model, increase the number of pulmonary metastases. Our findings uncover a novel non-receptor-mediated mechanism by which nanomaterials trigger intracellular signalling cascades via specific interaction with VE-cadherin, resulting in nanomaterial-induced endothelial cell leakiness.

A metabolomic study of low estimated GFR in non-proteinuric type 2 diabetes mellitus.

AIMS/HYPOTHESIS: We carried out a urinary metabolomic study to gain insight into low estimated GFR (eGFR) in patients with non-proteinuric type 2 diabetes. METHODS: Patients were identified as being non-proteinuric using multiple urinalyses. Cases (n = 44) with low eGFR and controls (n = 46) had eGFR values <60 and ≥60 ml min(-1) 1.73 m(-2), respectively, as calculated using the Modification of Diet in Renal Disease formula. Urine samples were analysed by liquid chromatography/mass spectrometry (LC/MS) and GC/MS. False discovery rates were used to adjust for multiple hypotheses testing, and selection of metabolites that best predicted low eGFR status was achieved using least absolute shrinkage and selection operator logistic regression. RESULTS: Eleven GC/MS metabolites were strongly associated with low eGFR after correction for multiple hypotheses testing (smallest adjusted p value = 2.62 × 10(-14), largest adjusted p value = 3.84 × 10(-2)). In regression analysis, octanol, oxalic acid, phosphoric acid, benzamide, creatinine, 3,5-dimethoxymandelic amide and N-acetylglutamine were selected as the best subset for prediction and allowed excellent classification of low eGFR (AUC = 0.996). In LC/MS, 19 metabolites remained significant after multiple hypotheses testing had been taken into account (smallest adjusted p value = 2.04 × 10(-4), largest adjusted p value = 4.48 × 10(-2)), and several metabolites showed stronger evidence of association relative to the uraemic toxin, indoxyl sulphate (adjusted p value = 3.03 × 10(-2)). The potential effect of confounding on the association between metabolites was excluded. CONCLUSIONS/INTERPRETATION: Our study has yielded substantial new insight into low eGFR and provided a collection of potential urinary biomarkers for its detection.

Plasma carotenoids and risk of acute myocardial infarction in the Singapore Chinese Health Study.

BACKGROUND AND AIM: Modification of low-density lipoprotein due to oxidative stress is essential in the development of coronary atherosclerosis. Data of specific carotenoids except ß-carotene on cardioprotective effects in humans are limited. METHODS AND RESULTS: This study examined the associations between plasma concentrations of specific carotenoids and incidence of acute myocardial infarction. The study included 280 incident cases of acute myocardial infarction and 560 matched controls nested within the Singapore Chinese Health Study, a prospective cohort of 63,257 Chinese men and women aged 45-74 years old enrolled in 1993-1998 in Singapore. Retinol and carotenoids in prediagnostic plasma were quantified using high-performance liquid chromatography. High levels of plasma ß-cryptoxanthin and lutein were associated with decreased risk of acute myocardial infarction after adjustment for multiple risk factors for coronary heart disease. For ß-cryptoxanthin, the odds ratio (95% confidence interval) for the highest (Q5) versus the lowest (Q1) quintile was 0.67 (0.37-1.21) (P for trend=0.03). For lutein, the odds ratios (95% confidence intervals) for the combined Q2-Q3 and the combined Q4-Q5 versus Q1 were 0.71 (0.45-1.12) and 0.58 (0.35-0.94) respectively (P for trend=0.03). There was no statistically significant association between other carotenoids or retinol and risk of acute myocardial infarction. CONCLUSIONS: High plasma levels of ß-cryptoxanthin and lutein were associated with decreased risk of acute myocardial infarction. The findings of this study support a cardioprotective role of these two carotenoids in humans.

A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress.

Under oxidative stress, poly(ADP-ribose) polymerase-1 (PARP-1) is activated and contributes to necrotic cell death through ATP depletion. On the other hand, oxidative stress is known to stimulate autophagy, and autophagy may act as either a cell death or cell survival mechanism. This study aims to explore the regulatory role of PARP-1 in oxidative stress-mediated autophagy and necrotic cell death. Here, we first show that hydrogen peroxide (H(2)O(2)) induces necrotic cell death in Bax-/- Bak-/- mouse embryonic fibroblasts through a mechanism involving PARP-1 activation and ATP depletion. Next, we provide evidence that autophagy is activated in cells exposed to H(2)O(2). More importantly, we identify a novel autophagy signaling mechanism linking PARP-1 to the serine/threonine protein kinase LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, leading to stimulation of autophagy. Finally, we demonstrate that autophagy plays a cytoprotective role in H(2)O(2)-induced necrotic cell death, as suppression of autophagy by knockdown of autophagy-related gene ATG5 or ATG7 greatly sensitizes H(2)O(2)-induced cell death. Taken together, these findings demonstrate a novel function of PARP-1: promotion of autophagy through the LKB1-AMPK-mTOR pathway to enhance cell survival in cells under oxidative stress.

Deformability study of breast cancer cells using microfluidics.

Cell deformability is an important biomarker which can be used to distinguish between healthy and diseased cells. In this study, microfluidics is used to probe the biorheological behaviour of breast cancer cells in an attempt to develop a method to distinguish between non-malignant and malignant cells. A microfabricated fluidic channel design consisting of a straight channel and two reservoirs was used to study the biorheological behaviour of benign breast epithelial cells (MCF-10A) and non-metastatic tumor breast cells (MCF-7). Quantitative parameters such as entry time (time taken for the cell to squeeze into the microchannel) and transit velocity (speed of the cell flowing through the microchannel) were defined and measured from these studies. Our results demonstrated that a simple microfluidic device can be used to distinguish the difference in stiffness between benign and cancerous breast cells. This work lays the foundation for the development of potential microfluidic devices which can subsequently be used in the detection of cancer cells.

AFM indentation study of breast cancer cells.

Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 degrees C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis.

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death.

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.

Reduced mitochondrial coenzyme Q10 levels in HepG2 cells treated with high-dose simvastatin: a possible role in statin-induced hepatotoxicity?

Lowering of low-density lipoprotein cholesterol is well achieved by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). Statins inhibit the conversion of HMG-CoA to mevalonate, a precursor for cholesterol and coenzyme Q10 (CoQ10). In HepG2 cells, simvastatin decreased mitochondrial CoQ10 levels, and at higher concentrations was associated with a moderately higher degree of cell death, increased DNA oxidative damage and a reduction in ATP synthesis. Supplementation of CoQ10, reduced cell death and DNA oxidative stress, and increased ATP synthesis. It is suggested that CoQ10 deficiency plays an important role in statin-induced hepatopathy, and that CoQ10 supplementation protects HepG2 cells from this complication.

Hypericin photoactivation triggers down-regulation of matrix metalloproteinase-9 expression in well-differentiated human nasopharyngeal cancer cells.

Recently, we have shown that hypericin-mediated photodynamic therapy (PDT) is a promising modality for the treatment of nasopharyngeal cancer (NPC). The present study evaluated the expression of matrix metalloproteinase-9 (MMP-9) following hypericin-PDT in well-differentiated HK1 NPC cells. Down-regulation of MMP-9 by hypericin-PDT was observed at the mRNA level in HK1 cells in vitro and in vivo and at the protein level in vitro. Transcriptional activities of the activator protein-1 (AP-1) and nuclear factor (NF)-kappaB regulatory elements were inhibited by PDT. We also found that PDT reduced secreted granulocyte-macrophage colony stimulating factor (GM-CSF), which is known to activate transcription of NK-kappaB and AP-1. However, incubation of untreated HK1 cells with exogenous GM-CSF abrogated the reduction of MMP-9 production in hypericin-PDT-treated cells. It would appear that PDT downregulates MMP-9 expression via inhibition of GM-CSF production, which in turn modulates AP1/NF-kappaB transcriptional activities. Suppression of MMP-9 by hypericin-PDT may have therapeutic implications.

Oxidative burden in prediabetic and diabetic individuals: evidence from plasma coenzyme Q(10).

AIM: Individuals with diabetes and prediabetes are at risk of vascular injury. However, the exact mechanisms are unclear. The mitochondria mobile electron carrier coenzyme Q(10) (CoQ(10)) is a potent lipophilic antioxidant. We hypothesize that oxidative stress, detectable as changes in plasma CoQ(10) concentrations and composition, plays an important role in vascular disease in diabetes. METHODS: We measured plasma CoQ(10) concentrations (including reduced ubiquinol and oxidized ubiquinone subfractions) in 60 subjects with normal glucose tolerance [NGT; fasting plasma glucose (FPG) < 5.5 mmol/l], 63 with impaired fasting glucose (IFG; FPG 5.6-6.9 mmol/l) and 69 with Type 2 diabetes (DM; FPG > 6.9 mmol/l). RESULTS: In men and women, the total CoQ(10)/total cholesterol ratio was reduced in DM (mean +/-sd) [male (M) 0.09 +/- 0.04; female (F) 0.07 +/- 0.04] compared with NGT (0.29 +/- 0.08; 0.21 +/- 0.07) and IFG (0.27 +/- 0.07; 0.23 +/- 0.07) (DM vs. NGT and IFG P = 0.001). A stepwise reduction in the plasma ubiquinol fraction (ubiquinol/total CoQ10) was observed from NGT (M 0.93 +/- 0.06; F 0.95 +/- 0.06) compared with IFG (0.43 +/- 0.25; 0.41 +/- 0.15) and DM (0.24 +/- 0.11; F 0.29 +/- 0.16) (DM vs. IFG vs. NGT P = 0.001). In contrast, the plasma ubiquinone/ubiquinol ratio increased from NGT (M 0.08 +/- 0.07, F 0.06 +/- 0.08) to IFG (2.14 +/- 1.84, 1.75 +/- 1.04) to DM (4.77 +/- 4.88, 3.81 +/- 3.71) (DM vs. IFG vs. NGT P = 0.001). These differences remained after adjusting for age, body mass index and FPG. CONCLUSIONS: The change in CoQ(10) with increasing FPG concentration suggests an increase in oxidative burden, already evident in the prediabetic IFG individuals. This increase in oxidative stress might contribute to the increased risk of vascular disease.

Increased uptake of divalent metals lead and cadmium into the brain after kainite-induced neuronal injury.

An increase in iron level, number of iron positive cells and ferritin expression has been observed in the rat hippocampus after neuronal injury induced by the excitotoxin, kainate. This is accompanied by an increased expression of divalent metal transporter-1 (DMT1) in the lesioned hippocampus, suggesting that the transporter may be partially responsible for the iron accumulation. DMT1 has a broad substrate range that includes other divalent metals such as lead (Pb) and cadmium (Cd), and the present study was carried out to elucidate the uptake of these metals in the kainate-injected brain. The technique of atomic absorption spectroscopy was used for analyses. Significantly higher lead and cadmium levels were detected in the hippocampus and other brain areas of intracerebroventricular kainate-injected rats treated with lead and cadmium in the drinking water, compared to intracerebroventricular saline-injected rats treated with lead and cadmium in the drinking water. Since very low levels of lead and cadmium are present in the normal animal, these results indicate increased uptake of lead and cadmium into brain areas as a result of the kainate injections. Increased iron levels were also detected in the hippocampus of the kainate-injected rats. The above results show increased uptake of divalent metals into brain areas undergoing neurodegeneration.

Emodin inhibits tumor cell migration through suppression of the phosphatidylinositol 3-kinase-Cdc42/Rac1 pathway.

Enhanced cell migration is one of the underlying mechanisms in cancer invasion and metastasis. Therefore, inhibition of cell migration is considered to be an effective strategy for prevention of cancer metastasis. We found that emodin (3-methyl-1,6,8-trihydroxyanthraquinone), an active component from the rhizome of Rheum palmatum, significantly inhibited epidermal growth factor (EGF)- induced migration in various human cancer cell lines. In the search for the underlying molecular mechanisms, we demonstrated that phosphatidylinositol 3-kinase (PI3K) serves as the molecular target for emodin. In addition, emodin markedly suppressed EGF-induced activation of Cdc42 and Rac1 and the corresponding cytoskeleton changes. Moreover, emodin, but not LY294002, was able to block cell migration in cells transfected with constitutively active (CA)-Cdc42 and CA-Rac1 by interference with the formation of Cdc42/Rac1 and the p21-activated kinase complex. Taken together, data from this study suggest that emodin inhibits human cancer cell migration by suppressing the PI3K-Cdc42/Rac1 signaling pathway.

Biological monitoring of kidney function among workers occupationally exposed to trichloroethylene.

AIMS: To investigate the nephrotoxic potential of trichloroethylene in a currently exposed population using sensitive urinary markers of kidney toxicity. METHODS: Renal dysfunction was monitored in a cross-sectional study of 70 workers currently exposed to trichloroethylene. An age and sex matched control population of 54 individuals was drawn from hospital and administrative staff. RESULTS: The mean exposure to trichloroethylene, estimated from urinary trichloroacetic acid concentrations, was 32 ppm (range 0.5-252 ppm) with an average duration of exposure of 4.1 years (range 1-20 years). Significant differences between the exposed and control populations were found for nephrotoxicity markers N-acetylglucosaminidase (NAG) and albumin, and for the mode of action marker, formic acid. However, neither NAG nor albumin showed a significant correlation with either the magnitude or duration of exposure to trichloroethylene. There was a significant correlation between urinary formic acid and trichloroacetic acid concentrations. Within the exposed population there were dose dependent increases in urinary methylmalonic acid concentrations and urinary glutathione S-transferase alpha activity. Although still within the control range, these changes were clearly dose dependent and consistent with one of the proposed mechanisms of trichloroethylene induced kidney toxicity. CONCLUSION: Although there was no evidence of kidney toxicity within the population studied, the results suggest that kidney damage could occur at exposure concentrations higher (>250 ppm) than those encountered in this study.

beta-Phenylethyl isothiocyanate-mediated apoptosis in hepatoma HepG2 cells.

beta-Phenylethyl isothiocyanate (PEITC) is a promising chemoprotective compound that is routinely consumed in the diet as its glucosinolate precursor. Previous studies have shown that PEITC can inhibit phase I enzymes and induce phase II detoxification enzymes along with apoptosis in vitro. The detailed mechanisms involved in the apoptotic cascade, however, have not been elucidated. In the present study, we demonstrate that PEITC can induce apoptosis in hepatoma HepG2 cells in a concentration- and time-dependant manner as determined by TUNEL positive and SubG1 population analysis. Caspase-3-like activity and poly(ADP-ribosyl)polymerase cleavage increased during treatment with 20 microM PEITC; high concentrations, however, induced necrosis. Pre-treatment with Z-VAD-FMK and the caspase-3-specific inhibitor Ac-DEVD-CHO prevented PEITC-induced apoptosis, as determined by caspase-3-like activity and DNA fragmentation. Additional investigations also showed that at concentrations of 5-10 microM PEITC, DNA synthesis was inhibited and G2/M phase cell cycle arrest occurred, correlating with an alteration in cyclin B1 and p34(cdc2) protein levels. Furthermore, we also demonstrate a concentration- and time-dependant burst of superoxide (O2*-) in PEITC-treated cells. However, pre- and co-treatment with the free radical scavengers Trolox, ascorbate, mannitol, uric acid and the superoxide mimetic manganese (III) tetrakis (N-methyl-2-pyridyl) porphyrin failed to prevent PEITC-mediated apoptosis. Taken together, these results suggest that PEITC potently induces apoptosis and cell cycle arrest in HepG2 cells and that the generation of reactive oxygen species appears to be a secondary effect.

Effluents from a pulp and paper mill: a skin and health survey of children living in upstream and downstream villages.

OBJECTIVES: A health survey of three villages (upstream village Rantau Baru and two downstream villages, Sering and Pelalawan) in the vicinity of a pulp and paper mill along the Kampar river in the province of Riau, Indonesia was conducted to find whether exposure to the effluents from the mill was related to skin conditions and ill health. METHODS: A cross sectional survey was carried out of children living in the three villages. RESULTS: Common skin conditions such as dermatitis, fungal infections, insect bites, and miliaria were found. No significantly increased risk of dermatitis or any illness in general was found with increasing levels of exposure to river water for downstream villages when compared with the upstream village. However, there was an increased risk of diarrhoea in Sering especially with drinking water directly from the river (prevalence rate ratio (PRR) 4.9, 95% confidence interval (95% CI) 0.4 to 63.9). An increased risk was also found within the upstream village Rantau Baru (PRR 2.3, 95% CI 0.9 to 5.8) and downstream village Sering (PRR 1.4, 95% CI 0.4 to 5.2) when children who drank water directly from the river were compared with those who never did. Analysis of the river water also showed physical and chemical variables within the acceptable range except for faecal coliforms (6 MPN/100 ml) found in the sample taken from Sering. CONCLUSIONS: The effluent from the mill is unlikely to be causing skin conditions and ill health. Diarrhoea may be due to faecal coliform contamination of the water because all raw sewerage is deposited in the river. Community health outreach programmes are being implemented based on these findings.

Critical role of reactive oxygen species formation in microcystin-induced cytoskeleton disruption in primary cultured hepatocytes.

Cyanobacteria (blue-green algae)-contaminated water is a worldwide public health problem. Microcystins are a group of liver-specific toxins generated by cyanobacteria. It is generally believed that the protein phosphorylation that leads to the disruption of intermediate filaments plays an important role in microcystin-induced hepatotoxicity. However, the mechanisms that contribute to the microcystin-induced alterations of microtubules and microfilaments are not fully understood. In the present study, the effects of microcystin-fR (M-LR), the most common microcystin, were examined on the organization of cellular microtubules and microfilaments in primary cultured rat hepatocytes. Our results indicate that M-LR initiated reactive oxygen species (ROS) formation followed by altering the cytoskeleton structures, which eventually led to significant LDH leakage. These effects were completely prevented by TEMPOL, a superoxide dismutase mimic, and also partially prevented by desferoxamine. These findings provide further evidence that ROS formation, especially superoxide radical, plays a crucial role in M-LR-induced disruption of cytoskeleton organization and consequent hepatotoxicity.

Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells.

Recent studies have demonstrated that induction of apoptosis is related to the cell growth inhibition potential of Salvia Miltiorrhiza (SM), a traditional herbal medicine. In the present study, we further explore the mechanistic pathway involved in SM-induced apoptosis in human hepatoma HepG2 cells. A rapid decline of intracellular glutathione (GSH) and protein thiol content was found in SM-treated cells. Moreover. SM exposure resulted in mitochondrial dysfunction as demonstrated by: (i) the onset of mitochondrial permeability transition (MPT); (ii) the disruption of mitochondrial membrane potential (MMP); and (iii) the release of cytochrome c from mitochondria into the cytosol. Subsequently, elevated level of intracellular reactive oxygen species (ROS) was observed prior to the onset of DNA fragmentation. However, no caspase-3 cleavage was observed throughout the whole period of SM treatment, while a caspase-3-independent poly(ADP-ribose) polymerase (PARP) cleavage was noted at the late stage in SM-induced apoptosis. Pretreatment of cells with N-acetylcysteine (NAC), the GSH synthesis precursor, conferred complete protection against MMP loss, ROS generation and apoptosis induced by SM. MPT inhibitors, cyclosporin A plus trifluoperazine, partially restored intracellular GSH content, and reduced SM-induced ROS formation and subsequently inhibited cell death. Moreover, antioxidants NAC, deferoxamine and catalase had little effect on GSH depletion and mitochondrial dysfunction, yet still were able to completely protect cells from SM-induced apoptosis. Taken together, our results suggest that SM deplete intracellular thiols, which, in turn, causes MPT and subsequent increase in ROS generation, and eventually apoptotic cell death.

A population-based prevalence survey of skin diseases in adolescents and adults in rural Sumatra, Indonesia, 1999.

Skin disorders are one of the most common presenting health problems and pose a considerable public health problem in developing and developed countries. A population-based survey (n = 917) was conducted in October and November 1999 to determine the prevalence of skin diseases in 3 rural villages in Indonesia (Riau province, Sumatra). The overall individual response rate was 96%. Household interviews were conducted to obtain information on skin conditions, basic socio-demographic factors and lifestyle habits. Trained doctors performed a thorough clinical skin examination of each subject in the household. The overall prevalence of skin disease in the 3 villages was 28.2% (95% confidence intervals 24.6-31.8). The prevalence of fungal infections (12.0%) was twice as high as dermatitis (5.1%). As skin conditions are common in rural Indonesia, it is advisable to improve the training of nurses and doctors in the prevention and treatment of skin disorders.

Cadmium-induced apoptosis and phenotypic changes in mouse thymocytes.

At present cadmium (Cd)-induced immunotoxicity and the mechanisms involved have not been fully elucidated. The main objective of the present study is to explore the apoptogenic property of Cd in primary cultured mouse thymocytes and its effect on cell surface marker expression and phenotypic changes. Cd-induced thymocyte apoptosis was determined by TdT-mediated dUTP nick end labeling (TUNEL) assay, DNA content/cell cycle analysis and DNA gel electrophoresis. The results showed that Cd was able to cause apoptosis in mouse thymocytes in a time- and dose-dependent manner. Moreover, different subsets of thymocytes possessed different susceptibility to the apoptotic effect of Cd, in the order of CD8+ > CD4- CD8- (double negative cells, DN) > CD4+ CD8+ (double positive cells, DP) > CD4+. Cd treatment also altered thymocyte surface marker expression, leading to evident phenotypic changes. Such changes were characterized by a decline in DP cells and a marked decrease in CD4+/CD8+ ratio, mainly due to a significant increase in CD8+ subsets. These observations help to obtain a better understanding of the immunotoxic and immunomodulatory effects of Cd.