The effect of 2,4-D on gene expression in cultured cells

The cytotoxic effects of exposure to low concentrations of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) that are typically found in groundwater were investigated, in vitro. Most 2,4-D toxicology studies use high concentrations of the herbicide that are above those typically found in groundwater and measure overt biological endpoints. In contrast, this thesis examines the effects of low concentrations of 2,4-D and measures more subtle and sensitive endpoints such as gene expression and the generation of reactive oxygen species. This work derives from recent cDNA microarray analysis conducted in our laboratory that revealed significant alterations in the expression of 238 genes in cells exposed to nanomolar (nM) concentrations of a commercial formulation of 2,4-D. These findings are extended in this thesis to include the in vitro cytotoxic effects of low concentrations of both technical and commercial 2,4-D on two cell lines. Cells derived from liver (HepG2) and kidney (HEK293) respectively, were chosen, since liver and kidney are known to metabolize 2,4-D in vivo. Cell viability was measured using the Resazurin assay, reactive oxygen species (ROS) were measured with 2’,7’-dichlorofluorescin diacetate (2’,7’-DCFH-DA), and real time–polymerase chain reaction (RT-PCR) was used to assess changes in mRNA expression while protein expression was examined by Western blot.Cell viability studies revealed that low environmental concentrations (0.1 to 100 nM) of 2,4-D induced small, but statistically significant decreases in cell viability. No concentration or time-dependent decreases in cell viability were observed in cells exposed to either forms of low environmental 2,4-D concentrations. HEK293 cells were more susceptible than HepG2 cells to the toxic effects of both forms of 2,4-D, having statistically significant lower viability at all exposure concentrations and durations. Both forms of 2,4-D reduced cell viability in both cell lines, suggesting that cytotoxicity was induced directly by 2,4-D, and not by the ‘inert ingredients’ in the commercial formulation.The ROS assays illustrated that 2,4-D induced statistically significant ROS production in HepG2 and HEK293 cell cultures at concentrations greater than 10 µM and 100 nM respectively. This was both a concentration and time-dependent effect in both cell lines. Although HEK293 cells were more susceptible to 2,4-D, they had 50 to 70% less ROS production than HepG2 cells, at all exposure concentrations and times.The RT-PCR and Western blot analyses showed that exposure of HepG2 and HEK293 cells to low 2,4-D concentrations induced (< 2 fold) alterations in mRNA and protein levels of FTL, FTH1 and PCNA however these changes did not consistently vary with concentration.Taken together, cell viability, ROS and gene expression studies show that low environmental 2,4-D concentrations induced subtle in vitro cytotoxic effects. However we have no evidence that these subtle changes pose a serious health threat to exposed humans

The effect of acyclovir on the tubular secretion of creatinine in vitro

<p>Abstract</p> <p>Background</p> <p>While generally well tolerated, severe nephrotoxicity has been observed in some children receiving acyclovir. A pronounced elevation in plasma creatinine in the absence of other clinical manifestations of overt nephrotoxicity has been frequently documented. Several drugs have been shown to increase plasma creatinine by inhibiting its renal tubular secretion rather than by decreasing glomerular filtration rate (GFR). Creatinine and acyclovir may be transported by similar tubular transport mechanisms, thus, it is plausible that in some cases, the observed increase in plasma creatinine may be partially due to inhibition of tubular secretion of creatinine, and not solely due to decreased GFR. Our objective was to determine whether acyclovir inhibits the tubular secretion of creatinine.</p> <p>Methods</p> <p>Porcine (LLC-PK1) and human (HK-2) renal proximal tubular cell monolayers cultured on microporous membrane filters were exposed to [2-¹⁴C] creatinine (5 μM) in the absence or presence of quinidine (1E+03 μM), cimetidine (1E+03 μM) or acyclovir (22 - 89 μM) in incubation medium.</p> <p>Results</p> <p>Results illustrated that in evident contrast to quinidine, acyclovir did not inhibit creatinine transport in LLC-PK1 and HK-2 cell monolayers.</p> <p>Conclusions</p> <p>The results suggest that acyclovir does not affect the renal tubular handling of creatinine, and hence, the pronounced, transient increase in plasma creatinine is due to decreased GFR, and not to a spurious increase in plasma creatinine.</p

New insight into Acyclovir Renal Handling and Nephrotoxicity

Drug – induced nephrotoxicity is a serious adverse reaction that can have deleterious effects on a patient’s health and well-being. Acyclovir is an example of such an agent that causes the aforesaid effects. The drug induces severe nephrotoxicity in patients. The etiology of acyclovir – induced nephrotoxicity has not been fully elucidated. The overall objective of this thesis is to gain new insight into the pathogenesis of acyclovir – induced nephrotoxicity. Cytotoxicity studies showed that acyclovir induced human renal proximal tubular (HK-2) cell death, in vitro, and that the degree of this toxicity was significantly reduced by co-exposure to 4-methylpyrazole. The results suggest that acyclovir induces direct insult to human renal proximal tubular cells and the toxicity may be caused by the parent drug’s noxious acyclovir aldehyde metabolite. Transepithelial transport studies illustrated that acyclovir does not inhibit the transport of creatinine across porcine renal proximal tubular (LLC-PK1) or HK-2 cell monolayers. The results suggest that acyclovir does not inhibit the tubular secretion of creatinine in vitro, and possibly, in vivo, as well. Therefore, the abrupt, pronounced and transient elevations in the levels of plasma creatinine observed in patients may be solely and genuinely due to reduced GFR as a result of acyclovir – induced nephrotoxicity, and not to a tubular interaction between creatinine and acyclovir. Employing human embryonic kidney cells (HEK293) containing the full-length human ABCG2 gene encoding the wildtype ABCG2 amino acid sequence; cell accumulation studies showed that in the presence of the human breast cancer resistance protein (BCRP) inhibitor, fumitremorgin C (FTC), there was significant intracellular accumulation of acyclovir. The results suggest that acyclovir is a substrate for the efflux transporter and bears several potential implications with respect to the renal transport mechanisms and pathogenesis of the direct tubular damage induced by the drug. Synthesizing all the data, the results contribute to a better understanding of the pathogenesis of acyclovir – induced nephrotoxicity. Moreover, the research highlights the need for future studies that will aid in further elucidation of the underlying cell and molecular mechanism(s) of this toxicity and potential therapies for prevention of the direct renal tubular injury induced by the drug.Ph

The effect of acyclovir on the tubular secretion of creatinine in vitro

Abstract Background While generally well tolerated, severe nephrotoxicity has been observed in some children receiving acyclovir. A pronounced elevation in plasma creatinine in the absence of other clinical manifestations of overt nephrotoxicity has been frequently documented. Several drugs have been shown to increase plasma creatinine by inhibiting its renal tubular secretion rather than by decreasing glomerular filtration rate (GFR). Creatinine and acyclovir may be transported by similar tubular transport mechanisms, thus, it is plausible that in some cases, the observed increase in plasma creatinine may be partially due to inhibition of tubular secretion of creatinine, and not solely due to decreased GFR. Our objective was to determine whether acyclovir inhibits the tubular secretion of creatinine. Methods Porcine (LLC-PK1) and human (HK-2) renal proximal tubular cell monolayers cultured on microporous membrane filters were exposed to [2-14C] creatinine (5 μM) in the absence or presence of quinidine (1E+03 μM), cimetidine (1E+03 μM) or acyclovir (22 - 89 μM) in incubation medium. Results Results illustrated that in evident contrast to quinidine, acyclovir did not inhibit creatinine transport in LLC-PK1 and HK-2 cell monolayers. Conclusions The results suggest that acyclovir does not affect the renal tubular handling of creatinine, and hence, the pronounced, transient increase in plasma creatinine is due to decreased GFR, and not to a spurious increase in plasma creatinine

3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity

Hepatic toxicology following single and multiple exposure of engineered nanomaterials utilising a novel primary human 3D liver microtissue model

BACKGROUND: The liver has a crucial role in metabolic homeostasis as well as being the principal detoxification centre of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated with accumulative production of nanomaterials, there is an urgent need to consider the possibility of detrimental health consequences of engineered NM exposure. It has been shown that exposure via inhalation, intratracheal instillation or ingestion can result in NM translocation to the liver. Traditional in vitro or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e. reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.). METHODS: In order to rectify these issues and for the very first time we have utilised a 3D human liver microtissue model to investigate the toxicological effects associated with a single or multiple exposure of a panel of engineered NMs (Ag, ZnO, MWCNT and a positively charged TiO(2)). RESULTS: Here we demonstrate that the repeated exposure of the NMs is more damaging to the liver tissue as in comparison to a single exposure with the adverse effects more significant following treatment with the Ag and ZnO as compared with the TiO(2) and MWCNT NMs (in terms of cytotoxicity, cytokine secretion, lipid peroxidation and genotoxicity). CONCLUSIONS: Overall, this study demonstrates that the human microtissue model utilised herein is an excellent candidate for replacement of traditional in vitro single cell hepatic models and further progression of liver nanotoxicology