Assessing consequences of marine pollution by hydrocarbons using sponges as model organisms

Pollution has been assessed as a mutagenic activity determined by the Ames test, using radiolabelled benzo[a]pyrene (BaP) as a model pollutant. Experimental animals were sponges, mainly Tethya lyncurium from the Northern Adriatic and from the Pacific near Catalina Island, California, U.S.A. Changes in ornithine decarboxylase (ODC) activity (ODC; EC 4.1.1.17) and polyamine concentrations with and without pollution were observed. There is a slow rise in ODC activity during the course of three-weeks exposure and a fast increase of polyamine levels during the course of one day. Mixed function oxygenase (MFO; EC 1.14) activity could not be detected in sponges. There was a significant concentration dependent coupling of radioactive BaP derivatives (BaPD) to the macromolecular fractions; the highest in protein, X 1000 greater than DNA and X 500 greater than RNA. Coupling is light-mediated and drops to zero in the dark. However when activated microsomal fractions from fish, that had been exposed to high level polycyclic aromatic hydrocarbon (PAH) pollution are added, dark incorporation rises to significant levels which can be decreased by the MFO inhibitor 7,8-benzoflavone (BP). The question of possible absence of DNA repair in the sponges and some implications are discussed.     

Toxic effects of some major polyaromatic hydrocarbons found in crude oil and aquatic sediments on Scenedesmus subspicatus

The green alga, Scenedesmus subspicatus was exposed for 7 days to a series of PAHs (polyaromatic hydrocarbons) of increased molecular weight from two to five rings [naphthalene (Nap), anthracene (Ant), phenanthrene (Phe), pyrene (Pyr) and benzo(a)pyrene (BaP)]. The toxicity measured as population growth inhibition by individual PAH to the S. subspicatus followed the order: BaP>Pyr>Ant>Phe>Nap. These results confirmed that the toxicity potential of PAHs seems to be strongly influenced by their physico-chemical properties (aqueous solubility, K(ow), coefficient of volatilization, etc.) and the conditions of algae culture (light, presence of nitrate ions, etc.). Consequently, Nap, Phe and Ant having low k(ow) values and low coefficient of volatilization values were less toxic than BaP with the highest k(ow) value, indicating for example why Nap with the lowest EC(50) value was nearly 2 x 10(5) times lower than that of BaP. Moreover, nitrate ions seemed to act directly on the degree of hydroxylated radical reactivity of PAHs, since BaP always remained the most toxic of the compounds tested. The results were also agreed with the QSAR model for toxicity prediction of PAHs to many aquatic organisms.     

Studies on oxidation of benzo [a] pyrene by sunlight and ozone

The destruction of the carcinogen benzo[a]pyrene (BaP) by light is well known; laboratory workers are routinely advised to cover fluorescent lamps with yellow filters while treating samples containing BaP. However until recently the mechanism of oxidation by sunlight and ozone had not been studied in detail. Concentrations of benzo[a]pyrene in urban air are in the range of 5–10 μg/1000 m³. Oxidant concentrations (predominantly in the form of ozone) are reported to be in the range of 0.01 ppm (22 μg per m³). Thus a sampling system with a filter paper would filter about 22,000 μg of ozone passing through and collect about 5 μg of BaP for analysis. The effect of interactions of such large ozone concentrations with BaP deposited on the filter paper is reported; data for the oxidation rates for benzo[a]pyrene coated on quartz surface and exposed to ozone or sunlight are presented. The oxidation products were analysed by thin-layer chromatography and highpressure liquid chromatography. From about eight products detected in these experiments, three have been identified as quinones based on UV-absorption spectrometry and mass spectrometry. Oxidation rates as high as 100% per hour of exposure are observed when less than 0.1 μg of BaP is coated inside the quartz tubes and exposed to ozone or sunlight. Oxidation rates for benzo[a]pyrene (using tritiated BaP) were determined for two types of experimental conditions. In the first set, tritiated BaP was spotted onto a glass fibre paper and sampling continued for 24 h; the loss of BaP was found to be 88%. In the second set tritiated BaP was spotted at intervals of one hour, for eight hours, while sampling is being carried out and the loss of BaP during the period was estimated to be 50%.     

DNA damage by benzo[a]pyrene in the liver of mosquito fish Gambusia affinis

Exposure of Gambusia affinis to water containing different concentrations of benzo[a]pyrene (BaP) causes an increase in benzo[a]pyrene monooxygenase (BPMO) activity which reaches a maximum on the second day. Concomitantly, the DNA is altered in such a way that nuclease S1-sensitive sites (SSS) become measurable. The size distribution of liver DNA treated with nuclease S1 in control fish shows two populations of DNA by length, with means of 30 X 10(6) and 60 X 10(6) Daltons, respectively. In fish treated with 100 ppb BaP, the population with longer molecules of DNA disappears and shorter molecules increase in number. This may be explained in terms of the introduction of an additional 0.31-0.46 DNA nicks per control DNA molecule caused by metabolically activated BaP derivatives.     

Histopathological evidence of antagonistic effects of tributyltin on benzo[a]pyrene toxicity in the Arctic charr (Salvelinus alpinus)

We previously reported that long-term (54 days), repeated intraperitoneal exposure to low doses of tributyltin (TBT; 0.3 mg/kg) inhibited the metabolic activation of co-administered benzo[a]pyrene (BaP; 3 mg/kg) in the Arctic charr (Salvelinus alpinus); BaP, in turn, stimulated the metabolism and/or excretion of TBT. Here, we report the results of histopathological examinations of liver, kidney and pseudobranch tissue samples originating from these same fish. The results revealed higher lesion incidences at all sampling time points (Days 8, 32 and 56) among BaP-exposed fish compared with fish exposed to either TBT alone or combined with BaP. The severity of lesions like necrosis was also higher in BaP-exposed fish. Moreover, hepatic basophilic foci were observed exclusively in fish exposed to BaP alone. Together, these results provide new evidences that TBT can antagonize BaP toxicity in fish exposed to both pollutants under controlled laboratory conditions. In contrast, BaP does not appear to provide protection against TBT toxicity.     

Evaluation of the noncovalent binding interactions between polycyclic aromatic hydrocarbon metabolites and human p53 cDNA

The binding of reactive polycyclic aromatic hydrocarbon (PAH) metabolites, formed enzymatically, to DNA is a crucial step in PAH carcinogenesis in vivo. We investigated the noncovalent binding interactions between 11 PAH metabolites and human p53 complementary DNA (p53 cDNA) using the fluorescence displacement method and molecular docking analysis. All of the examined metabolites predominantly interacted with p53 cDNA by intercalation instead of groove binding. The dissociation constants ranged from 0.02 to 12.34 μM. Of the metabolites tested, 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene showed the strongest binding affinities to DNA, while 2-naphthol was the weakest DNA intercalator. The intercalation of the metabolites was stabilized by stacking the PAH phenyl rings with the DNA base pairs and the formation of hydrogen bonds between the oxide or hydroxyl groups on the metabolites, and DNA bases or backbones. The binding of the metabolites to DNA showed some sequence selectivity. The binding affinities and hydrogen bonds for 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-4,5-dihydroepoxide (BPE) and benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE) differed. It seems that the functional groups on the periphery of the PAH aromatic ring play crucial roles in regulating its binding affinity with DNA. Although it was difficult to determine the correlation between DNA noncovalent binding affinity and carcinogenicity for some of the PAH metabolites, the present study improved our understanding of the formation of PAH metabolite-DNA adducts.     

Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide

We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24 d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T₉₉ (two-log reduction) of host-specific Bacteroidales cells was 28 h, whereas that of host-specific Bacteroidales DNA was 177 h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24 d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70 h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450 h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.     

Regenerating sponge cubes as a model in the impact evaluation of intermittant city and factory waste pollution

The siliceous sponge Geodia cydonium was induced to regenerate. In this condition the nucleic acid fractions were used as indicators to study the influence of pollution in marine environments. Besides conventional analytical methods a newly developed method is introduced. The cetyltrimethylammonium salts of the polyanions are formed which are soluble at high ionic strength and are precipitated sequentionally as measurable turbidities by dilution. The dilution turbidity curves of regenerating sponge in different environments aided with polyribosomal profiles can be interpreted. It is shown that under the conditions of intermittent pollution the amount of some high molecular RNA fractions decrease.The turbidity-dilution technique may become a useful tool in environmental investigations.     

Association of GSTM1 and GSTT1 polymorphisms with DNA damage in coal-tar workers

DNA damage was evaluated by alkaline comet assay in peripheral blood lymphocytes of 115 coal-tar workers occupationally exposed to polycyclic aromatic hydrocarbons (PAHs) and 105 control subjects. The effect of polymorphisms of glutathione S-transferase (GST) genotypes on the DNA damage was assessed. The mean tail moment (TM) value in the coal-tar workers was significantly higher as compared to the control subjects (12.06 ± 0.55 versus 0.44 ± 0.31; P < 0.05). No significant association (P > 0.05) between the GSTT1 and GSTM1 genotypes and the TM values was found, however highest mean rank TM value was reported in GSTM1 null and GSTT1 null genotypes in both control and exposed subjects. Our results suggest that there is increased DNA damage in coal-tar workers due to PAHs exposure. Polymorphisms in GSTM1 and GSTT1 genes do not show significant effect (P > 0.05) on DNA damage.     

Application of the k-ω turbulence model for a wind-induced vibration study of 2D bluff bodies

This paper is concerned with the question of an appropriate turbulence model for the computational modelling of bridge deck aero-elasticity. A detailed examination of the suitability of different turbulence models for simulating various aero-elastic phenomena leads to the conclusion that the two equation k-ω RANS turbulence model strikes the right balance between computational efficiency and accuracy in simulating the flow regime. In order to test this hypothesis a rectangular prism with B/D=4 is taken as an example structure and the flutter derivatives are identified from FSI simulations for both low and medium turbulent flow regimes. The simulations are carried out using a block iterative sequential coupling routine that allows for the exploitation of existing fluid and structural solvers. The results show that the k-ω can adequately model the motion induced shear layer dynamics that are necessary for simulating FSI. The results also demonstrate the potential benefits of computational FSI studies in that flutter derivatives (and other aerodynamic coefficients) can readily be obtained without some of the problems encountered in wind tunnel tests.     

A dynamic and complex monochloramine stress response in Escherichia coli revealed by transcriptome analysis

Despite the widespread use of monochloramine in drinking water treatment, there is surprisingly little information about its mode of action. In this study, DNA microarrays were used to investigate the global gene expression of Escherichia coli cells exposed to sub-lethal concentrations of monochloramine, with a focus on temporal dynamics. Genes induced by monochloramine were associated with several stress response functions, including oxidative stress, DNA repair, multidrug efflux, biofilm formation, antibiotic resistance, and cell wall repair. The diversity of functional associations supports a model of monochloramine action involving multiple cellular targets including cell membranes, nucleic acids, and proteins. These data suggest that E. coli responds to monochloramine exposure by activating diverse defense responses rather than a single antioxidant system and the exposure may also induce biofilm formation. The induction of multidrug efflux pumps and specific antibiotic resistance genes further suggests that exposure to monochloramine may contribute to reduced susceptibility to some antibiotics.     

Sub-zonal computational fluid dynamics in an object-oriented modelling framework

Airflow modelling is of fundamental importance for evaluating ventilation performance and energy consumption in buildings, and various approaches to the problem—starting from purely empirical up to the CFD ones—have been proposed and evaluated in the past years. Moreover, since the ultimate goal is whole building modelling, airflow simulation needs coupling with Energy Simulation (ES), in order to assess the overall energy performance. Due to the substantial differences between the software employed for airflow and ES, co-simulation is very often felt as the only way to handle such a problem. For example, in recent years a lot of effort has been spent in to couple ES and CFD tools. This paper proposes an alternative, in the form of an approach for solving the Navier-Stokes equations in a general multi-domain modelling framework. Since co-simulation is not involved, the correctness of the numerical solution relies on a single solver, thus being really transparent to the analyst. This is a first step towards a whole building simulation tool embedded in a unique framework capable of performing energy analysis, computing airflows, and representing control systems.     

Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC.     

Exposure to toxic agents alters organic elemental composition in human fingernails

Background and objective:

The human fingernail plate is highly keratinized. The keratin structure is held together by disulfide bonds. The organic elements sulfur and nitrogen occur almost exclusively in amino acids of the nail plate. This study analyzed whether occupational exposure to harmful chemical agents alters the organic elemental composition in fingernails.

Subjects and methods:

Participants were 71 occupationally exposed workers (49 hairdressers, 22 painters) and 49 unexposed controls. Aspects of exposure were assessed by questionnaire. Nail clippings of index and little fingers were analyzed for sulfur (S), nitrogen (N), carbon (C), and hydrogen (H) contents, using an automatic elemental analyzer (Vario EL III, Elementar Analysensysteme, Hanau, Germany). C/N, N/S, and C/S ratios were calculated. Reliability of elemental measurements was tested in a subset.

Results:

Reliability analysis of elemental measurements showed a very good agreement. Both painters and hairdressers displayed in their fingernails significantly lower percentages of sulfur, but higher percentages of carbon than unexposed controls. A trend was observed in that sulfur content was lowest in those hairdressers who reported structural changes in their fingernails. As a consequence, the C/S and N/S ratios were significantly increased in both exposed groups.

Conclusion:

The occupational use of harmful substances leads to decreased sulfur levels in the exposed persons, probably due to diminution of sulfur-rich proteins in the nails, resulting from destruction of disulfide-bonds by alkaline and acid groups. Thus, the C/S ratio seems to be a useful indicator for the amount of damage of nail protein by harmful agents.     

Control of biofilm formation in water using molecularly capped silver nanoparticles

Control of biofouling and its negative effects on process performance of water systems is a serious operational challenge in all of the water sectors. Molecularly capped silver nanoparticles (Ag-MCNPs) were used as a pretreatment strategy for controlling biofilm development in aqueous suspensions using the model organism Pseudomonas aeruginosa. Biofilm control was tested in a two-step procedure: planktonic P. aeruginosa was exposed to the Ag-MCNPs and then the adherent biofilm formed by the surviving cells was monitored by applying a model biofilm-formation assay. Under specific conditions, Ag-MCNPs retarded biofilm formation, even when high percentage of planktonic P. aeruginosa cells survived the treatment. For example, Ag-MCNPs (10 μg mL⁻¹) retarded biofilm formation (>60%), when 50 percent of the planktonic P. aeruginosa cells survived the treatment. Moreover, stable low value of relative biomass has been formed in the presence of fixed Ag-MCNPs concentrations at various biofilm incubation times. Our results showed that Ag-MCNPs pretreated cells were able to produce EPS although they succeeded to form relatively low adherent biofilm. These pretreated cells appear well preserved and undamaged under TEM HPH/freeze micrographs, yet the intra cellular material seems to be pushed towards the peripheral parts of the cell, possibly indicating a survival strategy to the presence of Ag-MCNPs. The lower value of relative biomass formed in the presence of Ag-MCNPs could be associated with molecular mechanisms related to biofilm formation or continuous release of silver ions in the sample. However, further research is required to examine these factors.     

Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: Implications for their use in assessing risk in recreational waters

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10 d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.     

Application of a molecular biology concept for the detection of DNA damage and repair during UV disinfection

As nucleic acids are major targets in bacteria during standardised UV disinfection (254 nm), inactivation rates also depend on bacterial DNA repair. Due to UV-related DNA modifications, PCR-based approaches allow for a direct detection of DNA damage and repair during UV disinfection. By applying different primer sets, the correlation between amplicon length and PCR amplification became obvious. The longer the targeted DNA fragment was, the more UV-induced DNA lesions inhibited the PCR. Regeneration of Pseudomonas aeruginosa, Enterococcus faecium, and complex wastewater communities was recorded over a time period of 66 h. While phases of intensive repair and proliferation were found for P. aeruginosa, no DNA repair was detected by qPCR in E. faecium. Cultivation experiments verified these results. Despite high UV mediated inactivation rates original wastewater bacteria seem to express an enhanced robustness against irradiation. Regeneration of dominant and proliferation of low-abundant, probably UV-resistant species contributed to a strong post-irradiation recovery accompanied by a selection for β-Proteobacteria.     

Speciation of lead, copper, zinc and antimony in water draining a shooting range—Time dependant metal accumulation and biomarker responses in brown trout (Salmo trutta L.)

The speciation of Pb, Cu, Zn and Sb in a shooting range run-off stream were studied during a period of 23 days. In addition, metal accumulation in gills and liver, red blood cell ALA-D activity, hepatic metallothionine (Cd/Zn-MT) and oxidative stress index (GSSG/ tGSH levels) in brown trout (Salmo trutta L.) exposed to the stream were investigated. Fish, contained in cages, were exposed and sampled after 0, 2, 4, 7, 9, 11 and 23 days of exposure. Trace metals in the water were fractionated in situ according to size (nominal molecular mass) and charge properties. During the experimental period an episode with higher runoff occurred resulting in increased levels of metals in the stream. Pb and Cu were mainly found as high molecular mass species, while Zn and Sb were mostly present as low molecular mass species. Pb, Cu and Sb accumulated on gills, in addition to Al origination from natural sources in the catchment. Pb, Cu and Sb were also detected at elevated concentration in the liver. Blood glucose and plasma Na and Cl levels were significantly altered during the exposure period, and are attributed to elevated concentrations of Pb, Cu and Al. A significant suppression of ALA-D was detected after 11 days. Significant differences were detected in Cd/Zn-MT and oxidative stress (tGSH/GSSG) responses at Day 4. For Pb the results show a clear link between the HMM (high molecular mass) positively charged Pb species, followed by accumulation on gills and liver and a suppression in ALA-D. Thus, high flow episodes can remobilise metals from the catchment, inducing stress to aquatic organisms.     

Electrochemical degradation of the β-blocker metoprolol by Ti/Ru 0.7 Ir 0.3 O 2 and Ti/SnO 2-Sb electrodes

Electrochemical oxidation has been proposed for the elimination of pesticides, pharmaceuticals and other organic micropollutants from complex waste streams. However, the detrimental effect of halide ion mediators and the generation of halogenated by-products in this process have largely been neglected thus far. In this study, we investigated the electrochemical oxidation pathways of the β-blocker metoprolol in reverse osmosis concentrate (ROC) from a water reclamation plant using titanium anodes coated with Ru_0.7Ir_0.3O₂ or SnO₂-Sb metal oxide layers. The results of liquid chromatography-mass spectrometry analysis indicated that irrespective of the electrode coating the same oxidant species participated in electrochemical transformation of metoprolol in ROC. Although Ti/SnO₂-Sb exhibited higher oxidizing power for the same applied specific electrical charge, the generation of large fractions of chloro-, chloro-bromo- and bromo derivatives was observed for both electrode coatings. However, degradation rates of metoprolol and its degradation products were generally higher for the Ti/SnO₂-Sb anode. Chemical analyses of metoprolol and its by-products were complemented with bioanalytical tools in order to investigate their toxicity relative to the parent compound. Results of the bioluminescence inhibition test with Vibrio fischeri and the combined algae test with Pseudokirchneriella subcapitata indicated a substantial increase in non-specific toxicity of the reaction mixture due to the formed halogenated by-products, while the specific toxicity (inhibition of photosynthesis) remained unchanged.     

Effect of chromium on the level of IL-12 and IFN-gamma in occupationally exposed workers

Chromium may affect humoral and cellular immunity, acting on T lymphocytes as well as on granulocytes and monocytes cells. Cytokines play an important role in the immune balance. In this study, the level of IL-12 and IFN-γ were evaluated in the sera and PHA/LPS stimulated culture supernatant of human PBMCs of healthy volunteers and occupationally exposed chromium workers. All the workers were highly exposed to chromium having mean of 104.65 ± 77.21 µg/dL (range 23.7-316.8 µg/dL). A suspension of exposed and unexposed human PBMC (0.5 × 10⁶ cells/ml) prepared and cultured in RPMI-1640 supplemented with 10% FCS for 18 h in the presence or absence of LPS (10 ng/ml) which used for stimulation of IL-12 and IFN-γ. The level of IL-12 and IFN-γ were evaluated in the sera samples as well as LPS stimulated and unstimulated culture supernatant of h-PBMCs of chromium exposed workers. In these chromium exposed workers the level of IL-12 was 433.66 ± 197.49 pg/ml and 983.45 ± 330.99 pg/ml in LPS stimulated culture supernatant of normal individuals and highly chromium exposed workers, which was significant (P < 0.05). Although the level of IL-12 was (78.61 ± 61.03 pg/ml to 146.52 ± 46.37 pg/ml) elevated in unstimulated culture supernatant of h-PBMCs of chromium exposed individuals as compared to control, but it was not significant. This observation also suggests that a significant increase in IFN-γ production in LPS stimulated and unstimulated culture supernatant of h-PBMCs of chromium exposed workers as compared to control. However, IFN-γ level have a significant positive correlation between blood chromium level (r = 0.833, t = 6.3872, P 0.05) and exposure time (in years) (r = 0.8916, t = 8.3540, P 0.05) of the occupationally exposed workers.