Differential protection and transactivation of P53, P21, Bcl2, PCNA, cyclin G, and MDM2 genes in rat liver and the HepG2 cell line upon exposure to pifithrin.

In response to genotoxic agents, normal tissue cells are instructed by p53 either to perform DNA repair or to undergo apoptosis. Studies showed that chemo and/or radiotherapy damage both normal and cancerous cells indiscriminately. To this end, severe side effects inflicted by p53 activation in normal tissues, would possibly be abrogated by p53 inhibition. Pifithrin-alpha (PFT-alpha) is a reversible inhibitor of p53-mediated apoptosis, p53-dependent gene transcription, as well as down stream responsive gene function. The objective of this study was (1) to evaluate PFT-alpha for differential cellular protection in response to arsenic trioxide and cadmium chloride exposure of normal and neoplastic cells, and (2) to evaluate the transcriptional activation of p53 and p53-responsive genes in rat liver cells and HepG2 carcinoma cell line. Cell survival was detected by fluorescein diacetate (FDA) and fluorospectroscopy. Mean LC50 and (SD) for HepG2 cells following exposure to arsenic were 13.7 (+/-1.0) microg/ml with PFT- alpha and 13.4 (+/- 0.5) microg/ml without PFT-alpha (p>0.05). For rat liver cells it was 670 (+/- 8.15) microg/ml with and 573.15 (+/-1.0) microg/ml without PFT-alphha (p<0.05). On exposure to cadmium Chloride, LC50's were 6.95 (+/-2.5) microg/ml for HepG2 cell line in presence of PFT-alpha and 7.35 (+/-1.9) microg/ml in its absence (p>0.5). The results revealed significant differences from controls only upon exposure of rat liver cells to arsenic trioxide in presence of PFT-alpha. PFT-alpha inhibited the transactivation of p53 in rat liver cells and resulted in repression of Bcl2, PCNA, MDM2, Cyclin G and P21 genes by arsenic trioxide. HepG2 cells exposed to arsenic trioxide and PFT-alpha showed expression of only the P53 and PCNA genes. We conclude that PFT-alpha exhibits cytoprotective effect, modifies the detrimental influences of known genotoxic agents in normal cells and has the potential for use as an adjuvant to cancer therapy.

Assessment of individual and combined toxicities of four non-essential metals (As, Cd, Hg and Pb) in the microtox assay.

Although most researches with non-essential metals (NEMs) have been done with single or individual metals, in reality, organisms are often exposed to multiple contaminants at the same time through the air, food and water. In this study, we tested the toxicity of four NEMs, As, Cd, Pb, and Hg, individually and as a composite mixture using the microtox bioassay. This assay uses the reduction of bioluminescence of the bacterium Vibrio fischeri as a measure of toxicity. The concentrations of each chemical in the mixture were based on multiples of their maximum contaminant levels (MCLs) set by the U.S. EPA. The highest concentration of exposure was 20 times the MCL, which translated into 200, 100, 40 and 300 ppb for As, Cd, Hg and Pb, respectively. The ratio for the mixture from these concentrations was 10:5:2:15 for As, Cd, Hg and Pb, respectively. Among the individual metals tested, the ranking of toxicity was Hg>Pb>Cd>As based on the EC50 values of 109, 455, 508 and 768 ppb for Hg, Pb, Cd and As, respectively. The EC50 for the composite mixture was 495% MCL which translated into nominal concentrations of 49, 25, 10 and 74 ppb for As, Cd, Hg, and Pb, respectively. Overall, the EC50 value of each NEM within the mixture was lower than the EC50 of the individual chemical; an evidence of synergism for the mixture. The individual toxic units (TU) were 0.06, 0.05, 0.09, and 0.16 for As, Cd Hg, and Pb, respectively and the summed toxic unit (TU) was 0.37 (less than 1). This study provides needed scientific data necessary for carrying out complete risk assessment of As, Cd, Hg, and Pb mixtures of some priority compounds.

A better method for standardizing vitellogenin content of fish tissues.

Vitellogenin (vtg) concentrations were measured in plasma and liver samples from 12 hybrid Tilapia oreochromis niloticus x O. aureus to compare concentrations in these tissues. The results were calculated under two different normalizations: volume per gram of sample used (similar to normalization usually published in the literature and typically used for ELISA) and volume per total protein (similar to normalization used in polyacrylamide gel electrophoresis; PAGE). It was observed that the normalization procedure used in PAGE (per gram total protein) minimized the method detection limit by about 1000 and 2500 times in plasma and liver respectively, compared to the normalization usually reported in the literature. It was also observed that normalizing per gram total protein makes it possible to eliminate a potential problem of accidental dilution of plasma samples during sample collection. Moreover, the normalization on a per gram of total protein makes it possible even to compare results from the two different methods namely PAGE and ELISA. It also allows comparison between different tissues. Using the normalization procedures as used in PAGE (per gram total protein) for liver and the normalization method as reported in literature for ELISA (per volume of sample used), it was observed that liver samples had higher vtg levels (mean: 62 microg vtg/g) compared to the corresponding plasma samples (mean: 0.24 microg vtg/ml). However, when both results were normalized per gram total protein all but one liver sample were lower (62 microg vtg/g) than the corresponding plasma concentrations (mean = 246 microg vtg/g).

Cytotoxicity of dinitrotoluenes (2,4-dNT, 2,6-DNT ) to MCF-7 and MRC-5 cells.

DNTs are considered possibly carcinogenic to humans (Group 2B) because there is inadequate evidence in humans for carcinogenicity though there is sufficient evidence in experimental animals. In this study, MCF-7 (breast) and MRC-5 (lung) cells were exposed to a serial dilution of 2,4 and 2,6 DNTs (control, 1-500 ppm) in 96 well tissue culture plates. After various time intervals (24, 48, 72 and 96 hrs) the plates were washed, and 100microl fluorescein diacetate solution (10 microg/ml in PBS) was added column wise to each well, and incubated at 37 C for 30 - 60 min before reading the fluorescence with a spectrofluorometer at excitation and emission wavelengths of 485 and 538 nm respectively. Spectrofluorometeric readings were converted to percentages of cell survival. Regression analysis was conducted to determine the relationship between cell survival and exposed concentration. Linear equations derived from the regression analysis were used to calculate the LC50 values. Results indicated that 2,6 DNT was more toxic to breast cells; LC50 values were 445 and 292 ppm at 24 and 48 hours respectively compared to 2,4 DNT showing LC50 values of 570 and 407 ppm at 24 and 48 hours, respectively. No significant differences in toxicity existed between the two chemicals with regard to lung cells. Contrary to the above observation, 2,4 DNT was more toxic to breast cells; LC50 values were 407 and 238 ppm at 24 and 48 hours respectively compared to lung cells showing LC50 values of 527 and 402 ppm at 24 and 48 hours respectively. No significant difference existed for 2,6 DNT between the two cell lines. Lungs cells were more resistant to the two chemicals.

Developmental arrest in Japanese medaka (Oryzias latipes) embryos exposed to sublethal concentrations of atrazine and arsenic trioxide.

Embryos of the Japanese medaka (Oryzias latipes) were exposed to serial concentrations of atrazine (0, 25, 50, and 100 ppm) and arsenic trioxide (0, 0.025, 0.05, 0.1 ppm) until hatching. Stasis of circulation, blood islands, titanic convulsions, tube heart and mortality were observed in atrazine-treated embryos. Each endpoint exhibited a concentration-response relationship. Only 4% of the embryos hatched in the 25 ppm, and none in the 50 and 100 ppm, probably due to cell death attributed to the embryos' inability to break from the chorion. With arsenic exposure, hatching was inversely correlated to chemical concentration: 86%, 75% and 54% for 0.025, 0.05 and 0.1 ppm, respectively. Hatching periods were also reduced from 7-13 days in controls to 7-11 days in arsenic-treated embryos. This observation was more pronounced with the 0.05 ppm concentration, showing a reduction of about 4 days. Despite this shortage in hatching time, there were no observable morphological abnormalities, as seen with atrazine. The ecological significance of these findings and implications for the development of sublethal toxicity tests using Japanese medaka embryos are important.

Lead-induced cytotoxicity and transcriptional activation of stress genes in human liver carcinoma (HepG2) cells.

Lead is a non-essential element that exhibits a high degree of toxicity, especially in children. Most research on lead has focused on its effects on organ systems such as the nervous system, the red blood cells, and the kidneys which are considered to be the primary targets of lead toxicity. However, the molecular mechanisms by which it induces toxicity, and carcinogenesis remain to be elucidated. In this research, we performed the MTT assay to assess the cytotoxicity, and the CAT-Tox assay to assess the transcriptional responses associated with lead exposure to thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2), by creating stable transfectants of mammalian promoter chloramphenicol (CAT) gene fusions. Study results indicated that lead nitrate is cytotoxic to HepG2 cells, showing LD50 values of 49.0 +/- 18.0 microg/mL, 37.5 +/- 9.2 microg/mL, and 3.5 +/- 0.7 microg/mL for cell mortality upon 24, 48 and 72 h of exposure, respectively; indicating a dose- and time-dependent response with regard to the cytotoxic effect of lead nitrate. A dose-response relationship was also recorded with respect to the induction of stress genes in HepG2 cells exposed to lead nitrate. Overall, six out of the thirteen recombinant cell lines tested showed inductions to statistically significant levels (p < 0.05). At 50 microg/mL of lead nitrate, the average fold inductions were: 2.1 +/- 1.0, 5.4 +/- 0.4, 12.1 +/- 6.2, 5.0 +/- 1.7, 2.5 +/- 1.3, and 4.8 +/- 4.5 for XRE, HSP70, CRE, GADD153, and GRP78, respectively. These results indicate the potential for lead nitrate to undergo biotransformation in the liver (XRE), to cause cell proliferation (c-fos), protein damage (HSP70, GRP78), metabolic perturbation (CRE), and growth arrest and DNA damage (GADD153). Marginal but not significant inductions were also obtained with the GSTYa (1.5 +/- 0.8), and GADD45 (5.7 +/- 8.1) promoters, and the NF-KB (2.0 +/- 1.7) response element, indicating the potential for oxidative stress. No significant inductions (p > 0.05) were recorded for CYP1A1, HMTIIA, p53RE, and RARE.

Pifithrin-alpha (PFT-alpha) caused differential protection of rat liver cells and HepG2 cell line in response to the selective cytotoxicity of arsenic and cadmium.

In response to genotoxic agents, normal cells are instructed by p53 to either perform DNA repair or to commit suicide. Since chemo and/or radiotherapy damage both normal and cancerous cells, the use of PFT-alpha, a reversible inhibitor of down stream function of p53, was suggested as a temporary inhibitor of p53-induced cell damage. Our objective therefore, was (1) to assess the inherent response of HepG2 and rat liver cells to the effects of arsenic and cadmium and (2) to evaluate the role of PFT-alpha in the differential protection of rat liver and HepG2 cells. Following cellular growth to 90% confluency, exposure to cytotoxic agents in presence of PFT-alpha (10 ppm) or its absence was performed. Cell survival was detected fluorometrically using fluorescein diacetate (FDA) and an Ascent Fluoroskan. Toxicity index (LC50) was calculated from percent survival using regression analysis. Results showed an average of 46 fold inherent resistance of rat liver cells to arsenic in comparison to HepG2 cells (LC50 range of 573.15-670 vs. 13.4-13.7 ppm respectively). An average of 8 fold inherent resistance was also attributed to rat liver cells in response to cadmium (LC50 range of 57.72-58.1 vs. 6.99-7.35 ppm respectively). PFT-alpha did not show significant difference in protecting HepG2 cells against cadmium or arsenic. In contrast, there was significant difference in the protection of rat liver cells upon exposure to arsenic. We conclude that Pifithrin-alpha exhibits protection to normal cells, which can play an important role in cancer chemotherapy.