Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions

This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of trafficking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via filter papers. The number of coelomocytes was significantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or significantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes.     

Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity

Seedlings of Theobroma cacao CCN 51 genotype were grown under greenhouse conditions and exposed to increasing concentrations of Cu (0.005, 1, 2, 4, 8, 16, and 32 mg Cu L^?1) in nutrient solution. When doses were equal or higher than 8 mg Cu L^?1, after 24 h of treatment application, leaf gas exchange was highly affected and changes in chloroplasts thylakoids of leaf mesophyll cells and plasmolysis of cells from the root cortical region were observed. In addition, cell membranes of roots and leaves were damaged. In leaves, 96 h after treatments started, increases in the percentage of electrolyte leakage through membranes were observed with increases of Cu in the nutrient solution. Moreover, there was an increase in the concentration of thiobarbituric acid-reactive substances in roots due to lipid peroxidation of membranes. Chemical analysis showed that increases in Cu concentrations in vegetative organs of T. cacao increased with the increase of the metal in the nutrient solution, but there was a greater accumulation of Cu in roots than in shoots. The excess of Cu interfered in the levels of Mn, Zn, Fe, Mg, K, and Ca in different organs of T. cacao. Analysis of gene expression via RTq-PCR showed increased levels of MT2b, SODCyt, and PER-1 expression in roots and of MT2b, PSBA, PSBO, SODCyt, and SODChI in leaves. Hence, it was concluded that Cu in nutrient solution at doses equal or above 8 mg L^?1 significantly affected leaf gas exchange, cell ultrastructure, and transport of mineral nutrients in seedlings of this T. cacao genotype.     

Stress-related gene expression changes in rainbow trout hepatocytes exposed to various municipal wastewater treatment influents and effluents

The present study sought to examine the performance of six different wastewater treatment processes from 12 wastewater treatment plants using a toxicogenomic approach in rainbow trout hepatocytes. Freshly prepared rainbow trout hepatocytes were exposed to increasing concentrations of influent (untreated wastewaters) and effluent (C₁₈) extracts for 48 h at 15 °C. A test battery of eight genes was selected to track changes in xenobiotic biotransformation, estrogenicity, heavy metal detoxification, and oxidative stress. The wastewaters were processed by six different treatment systems: facultative and aerated lagoons, activated sludge, biological aerated filter, biological nutrient removal, chemically assisted primary treated, and trickling filter/solids contact. Based on the chemical characteristics of the effluents, the treatment plants were generally effective in removing total suspended solids and chemical oxygen demand, but less so for ammonia and alkalinity. The 12 influents differed markedly with each other, which makes the comparison among treatment processes difficult. For the influents, both population size and flow rate influenced the increase in the following mRNA levels in exposed hepatocytes: metallothionein (MT), cytochrome P4503A4 (CYP3A4), and vitellogenin (VTG). Gene expression of glutathione S-transferase (GST) and the estrogen receptor (ER), were influenced only by population size in exposed cells to the influent extracts. The remaining genes—superoxide dismutase (SOD) and multidrug resistance transporter (MDR)—were not influenced by either population size or flow rate in exposed cells. It is noteworthy that the changes in MT, ER, and VTG in cells exposed to the effluents were significantly affected by the influents across the 12 cities examined. However, SOD, CYP1A1, CYP3A4, GST, and MDR gene expression were the least influenced by the incoming influents. The data also suggest that wastewater treatments involving biological or aeration processes had the best performance. We found that the effects of municipal effluents on gene expression depended on the population size, the initial properties of the incoming influent, and the wastewater treatment method applied. Considering that the long-term goals of wastewater treatment is to produce clean effluents for the aquatic biota and independent of the incoming influent, more research is needed in developing treatment processes to better protect aquatic life from anthropogenic contamination.     

Impairments of cadmium on vitellogenin accumulation in the hepatopancreas of freshwater crab <Emphasis Type="Italic">Sinopotamon henanense</Emphasis>

During ovary maturation of crabs, vitellogenin (Vg), a precursor molecule of vitellin (Vn) needed for embryogenesis, can be produced in large quantities in the hepatopancreas and then transported to the ovary by the hemolymph. In the present study, effects of Cd on Vg accumulation in the hepatopancreas and Vg transportation of the freshwater crab Sinopotamon henanense were investigated. We also studied the impacts of Cd on the mRNA expression of genes involved in energy metabolism, protein metabolism, and metallothionein (MT) and glutathione (GSH) synthesis. After Cd treatment, the Vg concentration and the Vg mRNA expression in the hepatopancreas were downregulated. Pearson’s correlation coefficient showed that the Vg level in the hepatopancreas correlated positively with those of the ovary and hemolymph (correlation coefficients 0.844 and 0.749, respectively), suggesting that the Vg transport from the hepatopancreas to the ovary can be impaired by Cd. The levels of carbohydrate and protein in the hepatopancreas of Cd-exposed crabs were decreased, and an inhibited protein metabolism was also observed. Energy production related isocitrate dehydrogenase and cytochrome C oxidase mRNA expressions, and MT and GSH synthesis increased after 10 days of Cd treatment and decreased after 20 days. Cd also caused a time-dependent upregulation of malondialdehyde. Our findings showed that Cd decreased Vg accumulation in the hepatopancreas due to partially excessive energy consumption and an activated defense system in the hepatopancreas, suggesting a possible regulatory mechanism in S. henanense which is the competitive advantage of energy reserves in metabolic Cd stress responses over the high-energy flux during vitellogenesis to ensure a continuous supply of metabolic energy. Moreover, the damage of Vg accumulation in the hepatopancreas caused by Cd could lead to an insufficient accumulation of Vn in the ovary and cause a retardation of oocyte development.     

Assessment of general condition of fish inhabiting a moderately contaminated aquatic environment

The assessment of general condition of fish in the moderately contaminated aquatic environment was performed on the European chub (Squalius cephalus) caught in September 2009 in the Sutla River in Croatia. Although increases of the contaminants in this river (trace and macro elements, bacteria), as well as physico-chemical changes (decreased oxygen saturation, increased conductivity), were still within the environmentally acceptable limits, their concurrent presence in the river water possibly could have induced stress in aquatic organisms. Several biometric parameters, metallothionein (MT), and total cytosolic protein concentrations in chub liver and gills were determined as indicators of chub condition. Microbiological and parasitological analyses were performed with the aim to evaluate chub predisposition for bacterial bioconcentration and parasitic infections. At upstream river sections with decreased oxygen saturation (～50 %), decreased Fulton condition indices were observed (FCI: 0.94 g?cm^?3), whereas gonadosomatic (GSI: 2.4 %), hepatosomatic (HSI: 1.31 %), and gill indices (1.3 %) were increased compared to oxygen rich downstream river sections (dissolved oxygen ～90 %; FCI: 1.02 g?cm^?3; GSI: 0.6 %; HIS: ～1.08 %; gill index: 1.0 %). Slight increase of MT concentrations in both organs at upstream (gills: 1.67 mg?g^?1; liver: 1.63 mg?g^?1) compared to downstream sites (gills: 1.56 mg?g^?1; liver: 1.23 mg?g^?1), could not be explained by induction caused by increased metal levels in the river water, but presumably by physiological changes caused by general stress due to low oxygen saturation. In addition, at the sampling site characterized by inorganic and fecal contamination, increased incidence of bacterial bioconcentration in internal organs (liver, spleen, kidney) was observed, as well as decrease of intestinal parasitic infections, which is a common finding for metal-contaminated waters. Based on our results, it could be concluded that even moderate contamination of river water by multiple contaminants could result in unfavourable living conditions and cause detectable stress for aquatic organisms.     

Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults

Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations.     

Assessing the hydrocarbon degrading potential of indigenous bacteria isolated from crude oil tank bottom sludge and hydrocarbon-contaminated soil of Azzawiya oil refinery,Libya

The disposal of hazardous crude oil tank bottom sludge (COTBS) represents a significant waste management burden for South Mediterranean countries. Currently, the application of biological systems (bioremediation) for the treatment of COTBS is not widely practiced in these countries. Therefore, this study aims to develop the potential for bioremediation in this region through assessment of the abilities of indigenous hydrocarbonoclastic microorganisms from Libyan Hamada COTBS for the biotreatment of Libyan COTBS-contaminated environments. Bacteria were isolated from COTBS, COTBS-contaminated soil, treated COTBS-contaminated soil, and uncontaminated soil using Bushnell Hass medium amended with Hamada crude oil (1 %) as the main carbon source. Overall, 49 bacterial phenotypes were detected, and their individual abilities to degrade Hamada crude and selected COBTS fractions (naphthalene, phenanthrene, eicosane, octadecane and hexane) were evaluated using MT2 Biolog plates. Analyses using average well colour development showed that ~90 % of bacterial isolates were capable of utilizing representative aromatic fractions compared to 51 % utilization of representative aliphatics. Interestingly, more hydrocarbonoclastic isolates were obtained from treated contaminated soils (42.9 %) than from COTBS (26.5 %) or COTBS-contaminated (30.6 %) and control (0 %) soils. Hierarchical cluster analysis (HCA) separated the isolates into two clusters with microorganisms in cluster 2 being 1.7- to 5-fold better at hydrocarbon degradation than those in cluster 1. Cluster 2 isolates belonged to the putative hydrocarbon-degrading genera; Pseudomonas, Bacillus, Arthrobacter and Brevundimonas with 57 % of these isolates being obtained from treated COTBS-contaminated soil. Overall, this study demonstrates that the potential for PAH degradation exists for the bioremediation of Hamada COTBS-contaminated environments in Libya. This represents the first report on the isolation of hydrocarbonoclastic bacteria from Libyan COTBS and COTBS-contaminated soil.     

Characterization and cadmium-resistant gene expression of biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11

Biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11 was isolated from coastal marine sediment of Paradeep Port, Odisha, East Coast, India, which resisted up to 1,000 ppm of cadmium (Cd) as cadmium chloride in aerobic conditions with a minimal inhibitory concentration of 1,250 ppm. Biomass and extracellular polymeric substances (EPS) secreted by the cells effectively removed 58.760?±?10.62 and 29.544?±?8.02 % of Cd, respectively. The integrated density of the biofilm-EPS observed under fluorescence microscope changed significantly (P?≤?0.05) in the presence of 50, 250, 450, 650 and 850 ppm Cd. ATR-FTIR spectroscopy showed a peak at 2,365.09/cm in the presence of 50, 250, 450 and 650 ppm Cd which depicts the presence of sulphydryl group (–SH) within the EPS, whereas, a peak shift to 2,314.837/cm in the presence of 850 ppm Cd suggested the major role of this functional group in the binding with cadmium. On exposure to Cd at 100, 500 and 1,000 ppm, the expression profiles of cadmium resistance gene (czcABC) in the isolate showed an up-regulation of 3.52-, 17- and 24-fold, respectively. On the other hand, down-regulation was observed with variation in the optimum pH (6) and salinity (20 g l^?1) level. Thus, the cadmium resistance gene expression increases on Cd stress up to the tolerance level, but an optimum pH and salinity are the crucial factors for proper functioning of cadmium resistance gene.     

Adaptation of the Daphnia sp. acute toxicity test: miniaturization and prolongation for the testing of nanomaterials

Manufacturing of nanomaterials (NMs) is often complex and expensive, and their environmental risks are poorly understood or even unknown. An economization of testing NMs is therefore desirable, which can be achieved by miniaturizing test systems. However, the downsizing of test vessels and volumes can enlarge the surface/volume ratio (SVR) which in turn can affect the bioavailable concentration of adsorbing substances like NMs. The present study focused on the miniaturization of the acute toxicity test with Daphnia magna. The adaptations were verified with three reference substances, the non-adsorbing potassium dichromate (K₂Cr₂O₇) and as potentially highly-adsorbing substances silver nanoparticles (AgNPs) and silver nitrate (AgNO₃). The miniaturized test was conducted in 24-well microtiter plates (MT) and simultaneously compared to the OECD standard test (ST). Furthermore, the test duration was prolonged from 48 to 96 h since NMs tend to show effects only after extended exposure. The toxicity of K₂Cr₂O₇ and AgNPs continued to increase within the prolonged test span. The test comparisons with K₂Cr₂O₇ did not reveal any significant differences between ST and MT. AgNO₃ toxicity was significantly decreased in MT compared to ST due to the enlarged SVR. The toxicity of AgNPs in MT after 24 h was equal to ST. Contrary to our expectations an exposure longer than 24 h resulted in an increase of AgNP toxicity in MT, possibly due to enhanced dissolution of silver. Microtiter plates are appropriate alternative test vessels for the Daphnia sp. acute toxicity test; thus, its miniaturization is feasible. The enlarged SVR has to be taken into account since it can affect the toxicity of potentially adsorbing substances. Furthermore, the standard test duration of 48 h might underestimate the toxicity of many substances, especially of NMs.     

Effects of allelochemical extracted from water lettuce (Pistia stratiotes Linn.) on the growth, microcystin production and release of Microcystis aeruginosa

This study explored the optimisation of a method of extracting allelochemicals from Pistia stratiotes Linn., identified the optimal dose range for the allelochemicals’ anti-algal effect and investigated their impact on the growth of Microcystis aeruginosa, as well as the production and release of microcystin-LR (MC-LR). Based on measured changes in algal cell density and chlorophyll a (Chl-a) content, the allelochemicals were confirmed to have the strongest anti-algal effect with the lowest half-effect concentration of 65 mg L^?1 when they were extracted using ethyl acetate as the extraction solvent, 1:20 g mL^?1 as the extraction ratio and 1 h as the extraction time. The allelochemicals extracted from P. stratiotes using this optimal method exhibited the strongest inhibitory effect on the growth of algae when used within a dose range of 60–100 mg L^?1; the relative inhibitory ratio reached 50–90 %, and Chl-a content reduced 50–75 % in algae cell cultures within 3–7 days. In addition, the extracted allelochemical compounds demonstrated no significant impact on the extracellular release of MC-LR during the culturing period. The amount of intracellular MC-LR per 10⁶ algal cells increased depending on the increasing dose of allelochemicals from P. stratiotes after 7 days of culturing and maintained stability after 16 days. There was no increase in the total amount of MC-LR in the algal cell culture medium. Therefore, the application of allelochemicals from P. stratiotes to inhibit M. aeruginosa has a high degree of ecological safety and can be adopted in practical applications for treating water subjected to algae blooms because the treatment can effectively inhibit the proliferation of algal cells without increasing the release of cyanotoxin.     

Differential expression profile of membrane proteins in Aplysia pleural–pedal ganglia under the stress of methyl parathion

This study was aimed to analyze the alteration of membrane protein profiles in Aplysia juliana Quoy & Gaimard (A. juliana) pleural–pedal ganglia under MP exposure. Both the results of GC–MS analysis and the activity assay of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT) reveal that MP toxicological effects on Aplysia left and right pleural–pedal ganglia are different under 7 and 14 days of exposure. Therefore, Aplysia were subjected for exposure at two concentrations (1 and 2 mg/l) of MP for 7 and 14 days for membrane proteomic study. As a result, 19 and 14 protein spots were differentially expressed in A. juliana left pleural–pedal ganglia under 7 and 14 days treatment, and 20 and 14 protein spots found with differential expressions in their right ganglia under the same treatment, respectively. Several proteins with expression variations were detected from both the left and right pleural–pedal ganglia; however, most proteins have distinctive expressions, indicating different mechanisms might be involved in initiating MP toxicology in left and right ganglia. Among the total differential protein spots obtained, 29 proteins were classed as membrane proteins. These proteins are mainly involved in the metabolism process, cell redox homeostasis, signal transduction, immunology, intracellular transport and catalysis, indicating MP toxicity in mollusks seems to be complex and diverse. Some differentially expressed proteins were further confirmed by Western blotting and quantitative real-time PCR. These results might provide renovated insights to reveal the mechanism of MP-induced neurotoxicity, and the novel candidate biomarkers might have potential application for environmental evaluation of MP pollution level.     

Molecular cloning and expression of novel metallothionein (MT) gene in the polychaete Perinereis nuntia exposed to metals

To report a novel metallothionein (MT) gene and evaluate its potency as a biomarker, we clone this MT gene and measured the expression levels in the metal-exposed polychaete Perinereis nuntia. Accumulated metal contents and metallothionein-like proteins (MTLPs), which have been recognized as potential biomarkers, were compared with the relative mRNA expressions of the MT gene of P. nuntia (Pn-MT). In addition, the metal-binding affinity was estimated by recombinant Pn-MT protein. Pn-MT having high cysteine residues with three metal response elements in the promoter region closely clusters with those of other invertebrates. The accumulation patterns of metals were dependent on the exposure times in lead (Pb), cadmium (Cd), and copper (Cu) exposure. Particularly, both MTLP levels and relative mRNA expressions of MT were increased with accumulated metal contents and exposure time in P. nuntia exposed to Pb and Cd. There was no significant modulation of the Pn-MT gene in polychaetes exposed to Zn and As. However, the metal-binding ability of the recombinant Pn-MT protein provides a clear evidence for a high affinity of MT to several metal elements. These results suggest that Pn-MT would play an important role in the detoxification and/or sequestration of specific metals (e.g., Pb and Cd) in P. nuntia and have potential as a molecular biomarker in the monitoring of the marine environment using a polychaete.     

Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum

Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.     

Is the microbiological quality of the Msunduzi River (KwaZulu-Natal, South Africa) suitable for domestic, recreational, and agricultural purposes?

As little is known about the potential risks associated with the use of microbiologically contaminated river water for recreation, irrigation, or domestic purposes, the Msunduzi River in Pietermaritzburg (KwaZulu-Natal, South Africa) was evaluated. In addition to pH, temperature, and chemical oxygen demand, quantitative and qualitative microbiological analyses were performed monthly for 13 months. These included aerobic plate counts, counts of aerobic and anaerobic sporeformers, most probable numbers for total and faecal coliforms and Escherichia coli and the detection of Salmonella spp., Staphylococcus aureus, and intestinal enterococci. Presumptive E. coli and S. aureus from river water samples were confirmed using PCR and additionally matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) for E. coli. Aerobic plate counts were above the South African Department of Water Affairs recommended guideline level for domestic use of 100 cfu/ml for all 13 months assessed. Faecal coliform (up to 63,000 MPN/100 ml) and E. coli (up to 7,900 MPN/100 ml) levels regularly exceeded stipulated limits for safe irrigation, domestic and recreational use. The presence of Salmonella spp., S. aureus, and intestinal enterococci frequently coincided with faecal coliform and E. coli levels above 1,000 MPN/100 ml. This illustrates the value of using guideline values for faecal coliforms and E. coli as indicators for the presence of potential pathogens. PCR and MALDI-TOF MS confirmation of E. coli were in agreement, thereby demonstrating the potential of MALDI-TOF MS as a suitable alternative. These data demonstrate that potential health risks are associated with using Msunduzi River water for irrigation and recreational or domestic purposes.     

Evaluating the phytoremediation potential of Phragmites australis grown in pentachlorophenol and cadmium co-contaminated soils

Pot-culture experiments were conducted to evaluate the phytoremediation potential of a wetland plant species, Phragmites australis in cadmium (Cd) and pentachlorophenol (PCP) co-contaminated soil under glasshouse conditions for 70 days. The treatments included Cd (0, 5 and 50 mg kg^?1) without or with PCP (50 and 250 mg kg^?1). The results showed that growth of P. australis was significantly influenced by interaction of Cd and PCP, decreasing with either Cd or PCP additions. Plant biomass was inhibited and reduced by the rate of 89 and 92 % in the low and high Cd treatments and by 20 and 40 % in the low and high PCP treatments compared to the control. The mixture of low Cd and low PCP lessened Cd toxicity to plants, resulting in improved plant growth (by 144 %). Under the joint stress of the two contaminants, the ability of Cd uptake and translocation by P. australis was weak, and the BF and TF values were inferior to 1.0. A low proportion of the metal is found aboveground in comparison to roots, indicating a restriction on transport upwards and an excluding effect on Cd uptake. Thus, P. australis cannot be useful for phytoextraction. The removal rate of PCP increased significantly (70 %) in planted soil. Significant positive correlations were found between the DHA and the removal of PCP in planted soils which implied that plant root exudates promote the rhizosphere microorganisms and enzyme activity, thereby improving biodegradation of PCP. Based on results, P. australis cannot be effective for phytoremediation of soil co-contaminated with Cd and PCP. Further, high levels of pollutant hamper and eventually inhibit plant growth. Therefore, developing supplementary methods (e.g. exploring the partnership of plant–microbe) for either enhancing (phytoextraction) or reducing the bioavailability of contaminants in the rhizosphere (phytostabilization) as well as plant growth promoting could significantly improve the process of phytoremediation in co-contaminated soil.     

Increasing CACNA1C expression in placenta containing high Cd level: an implication of Cd toxicity

Cadmium (Cd) has known to produce many adverse effects on organs including placenta. Many essential transporters are involved in Cd transport pathways such as DMT-1, ZIP as well as L-VDCC. Fourteen pregnant women participated and were divided into two groups: high and low Cd-exposed (H-Cd, L-Cd) groups on the basis of their residential areas, Cd concentrations in the blood (B-Cd), urine (U-Cd), and placenta (P-Cd). The results showed that the B-Cd and U-Cd were significantly increased in H-Cd group (p < 0.05). Interestingly, the P-Cd in H-Cd group was elevated (p < 0.05) and positively related to their B-Cd and U-Cd values (p < 0.05). However, the mean cord blood Cd (C-Cd) concentration in H-Cd group was not significantly increased about 2.5-fold when comparing to L-Cd group. To determine the Cd accumulation in placental tissues, metallothionein-1A (MT-1A) and metallothionein-2A (MT-2A) expressions were used as biomarkers. The results revealed that mean MT-1A and MT-2A mRNAs and MT-1/2 proteins were up-regulated in H-Cd group (p < 0.05). In addition, the Ca channel alpha 1C (CACNA1C) mRNA and protein expressions were noticeably elevated in H-Cd group (p < 0.05). From these findings, we suggested that CACNA1C might be implicated in Cd transport in human placenta.