Frederick A. Beland

Malachite Green: A Toxicological Review

Malachite green, an N-methylated diaminotriphenylmethane dye, is used primarily as a therapeutic agent in aquaculture. In solution, the dye exists as a mixture of the cation (chromatic malachite green) and its carbinol base, with the ratio depending on the pH of the solution; the dye also can undergo chemical and metabolic reduction to a leuco derivative. Analysis offish tissue after exposure to malachite green indicates the presence of both chromatic and leuco forms, with the latter having a much longer tissue half-life. Malachite green intercalates with DNA, with a preference for A:T-rich regions, and the leuco derivative bears a structural resemblance to carcinogenic aromatic amines that can form covalent DNA adducts. Malachite green is mutagenic in Salmonella typhimurium TA98 in the presence of an exogenous metabolizing system. In mammalian cells, it shows marked cytotoxicity and the ability to induce cell transformation and lipid peroxidation. Results from carcinogenicity bioassays with malachite green have been equivocal; however, it appears to act as a tumor promoter, perhaps because of its ability to induce the formation of reactive oxygen species. These characteristics, plus its close structural similarity to carcinogenic triphenylmethane dyes (e.g., gentian violet) suggest that additional data are required to determine if human exposure to malachite green results in adverse health effects.

An Evaluation of the Biological and Toxicological Properties of Aloe Barbadensis (Miller), Aloe Vera

Aloe barbadensis (Miller), Aloe vera, has a long history of use as a topical and oral therapeutic. The plant is the source of two products, gel and latex, which are obtained from its fleshy leaves. Aloe vera products contain multiple constituents with potential biological and toxicological activities, yet the active components elude definition. Ingestion of Aloe vera is associated with diarrhea, electrolyte imbalance, kidney dysfunction, and conventional drug interactions; episodes of contact dermatitis, erythema, and phototoxicity have been reported from topical applications. This review examines the botany, physical and chemical properties, and biological activities of the Aloe vera plant.

E-cadherin transcriptional down-regulation by epigenetic and microRNA-200 family alterations is related to mesenchymal and drug-resistant phenotypes in human breast cancer cells

The conversion of early stage tumors into invasive malignancies with an aggressive phenotype has been associated with the irreversible loss of E‐cadherin expression. The loss of E‐cadherin expression in human tumors, including breast cancer, has been attributed to promoter CpG island hypermethylation and direct inhibition by transcriptional repressors. Recent evidence demonstrates that up‐regulation of E‐cadherin by microRNA‐200b (miR‐200b) and miR‐200c through direct targeting of transcriptional repressors of E‐cadherin, ZEB1, and ZEB2, inhibits epithelial‐to‐mesenchymal transition (EMT), a crucial process in the tumor progression. We demonstrate that microRNA miR‐200 family‐mediated transcriptional up‐regulation of E‐cadherin in mesenchymal MDA‐MB‐231 and BT‐549 cells is associated directly with translational repression of ZEB1 and indirectly with increased acetylation of histone H3 at the E‐cadherin promoter. The increase in histone H3 acetylation may be attributed to the disruption of repressive complexes between ZEB1 and histone deacetylases and to the inhibition of SIRT1, a class III histone deacetylase. These events inhibit EMT and reactivate a less aggressive epithelial phenotype in cancer cells. Additionally, disruption of ZEB1‐histone deacetylase repressor complexes and down‐regulation of SIRT1 histone deacetylase up‐regulate proapoptotic genes in the p53 apoptotic pathway resulting in the increased sensitivity of cancer cells to the chemotherapeutic agent doxorubicin.

DNA hypomethylation in the origin and pathogenesis of human diseases

The pathogenesis of any given human disease is a complex multifactorial process characterized by many biologically significant and interdependent alterations. One of these changes, specific to a wide range of human pathologies, is DNA hypomethylation. DNA hypomethylation signifies one of the major DNA methylation states that refers to a relative decrease from the “normal” methylation level. It is clear that disease by itself can induce hypomethylation of DNA; however, a decrease in DNA methylation can also have an impact on the predisposition to pathological states and disease development. This review presents evidence suggesting the involvement of DNA hypomethylation in the pathogenesis of several major human pathologies, including cancer, atherosclerosis, Alzheimer’s disease, and psychiatric disorders.

Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice

Malachite green, an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in commercial fish hatcheries. Malachite green is reduced to and persists as leucomalachite green in the tissues of fish. Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm malachite green or 1160 ppm leucomalachite green for 28 days to determine the toxicity and metabolism of the dyes. Apoptosis in the transitional epithelium of the urinary bladder occurred in all mice fed the highest dose of leucomalachite green. This was not observed with malachite green. Hepatocyte vacuolization was present in rats administered malachite green or leucomalachite green. Rats given leucomalachite green also had apoptotic thyroid follicular epithelial cells. Decreased T4 and increased TSH levels were observed in male rats given leucomalachite green. A comparison of adverse effects suggests that exposure of rats or mice to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. N-Demethylated and N-oxidized malachite green and leucomalachite green metabolites, including primary arylamines, were detected by high performance liquid chromatography/mass spectrometry in the livers of treated rats. 32P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA. These data suggest that malachite green and leucomalachite green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to malachite green.

Difference in expression of hepatic microRNAs miR-29c, miR-34a, miR-155, and miR-200b is associated with strain-specific susceptibility to dietary nonalcoholic steatohepatitis in mice

The importance of dysregulation of microRNA (miRNA) expression in nonalcoholic steatohepatitis (NASH) has been increasingly recognized; however, the association between altered expression of miRNAs and pathophysiological features of NASH and whether there is a connection between susceptibility to NASH and altered expression of miRNAs are largely unknown. In this study, male inbred C57BL/6J and DBA/2J mice were fed a lipogenic methyl-deficient diet that causes liver injury similar to human NASH, and the expression of miRNAs and the level of proteins targeted by these miRNAs in the livers were determined. Administration of the methyl-deficient diet triggered NASH-specific changes in the livers of C57BL/6J and DBA/2J mice, with the magnitude being more severe in DBA/2J mice. This was evidenced by a greater extent of expression of fibrosis-related genes in the livers of methyl-deficient DBA/2J mice. The development of NASH was accompanied by prominent changes in the expression of miRNAs, including miR-29c, miR-34a, miR-155, and miR-200b. Interestingly, changes in the expression of these miRNAs and protein levels of their targets, including Cebp-β, Socs 1, Zeb-1, and E-cadherin, in the livers of DBA/2J mice fed a methyl-deficient diet were more pronounced as compared with those in C57BL/6J mice. These results show that alterations in the expression of miRNAs are a prominent event during development of NASH induced by methyl deficiency and strongly suggest that severity of NASH and susceptibility to NASH may be determined by variations in miRNA expression response. More important, our data provide a mechanistic link between alterations in miRNA expression and pathophysiological and pathomorphological features of NASH.

Down-regulation of the microRNAs miR-34a, miR-127, and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet†

Altered expression of microRNAs (miRNAs) has been reported in diverse human cancers; however, the down‐regulation or up‐regulation of any particular miRNAs in cancer is not sufficient to address the role of these changes in carcinogenesis. In this study, using the rat model of liver carcinogenesis induced by a methyl‐deficient diet, which is relevant to the hepatocarcinogenesis in humans associated with viral hepatitis C and B infections, alcohol exposure and metabolic liver diseases, we showed that the development of hepatocellular carcinoma (HCC) is characterized by prominent early changes in expression of miRNA genes, specifically by inhibition of expression of microRNAs miR‐34a, miR‐127, miR‐200b, and miR‐16a involved in the regulation of apoptosis, cell proliferation, cell‐to‐cell connection, and epithelial‐mesenchymal transition. The mechanistic link between these alterations in miRNAs expression and the development of HCC was confirmed by the corresponding changes in the levels of E2F3, NOTCH1, BCL6, ZFHX1B, and BCL2 proteins targeted by these miRNAs. The significance of miRNAs expression dysregulation in respect to hepatocarcinogenesis was confirmed by the persistence of these miRNAs alterations in the livers of methyl‐deficient rats re‐fed a methyl‐adequate diet. Altogether, the early occurrence of alterations in miRNAs expression and their persistence during the entire process of hepatocarcinogenesis indicate that the dysregulation of microRNAs expression may be an important contributing factor in the development of HCC.

Rapid isolation of carcinogen-bound DNA and RNA by hydroxyapatite chromatography

Carcinogen-bound DNA and RNA are conveniently isolated by solvent extraction and hydroxyapatite (HAP) chromatography. Tissue is suspended in 8 M urea-0.24 M sodium phosphate-1% sodium dodecyl sulfate-10 mM EDTA, pH 6.8 (MUP-SDS-EDTA) and extracted with chloroform-isoamyl alcohol-phenol (24:1:25; CIP) to remove protein. RNA and DNA are separated by passing the aqueous solution through an HAP column; RNA is eluted with MUP, DNA with 0.48 M sodium phosphate, pH 6.8. Examples presented are: (1) calf thymus DNA that has been reacted with N-acetoxy-2-acetylaminofluorene (N-OAc-AAF), (2) isolated intact rat hepatocytes incubated with N-hydroxy-AAF and (3) livers from Sprague-Dawley rats treated with N-hydroxy-AAF.

Methods of DNA adduct determination and their application to testing compounds for genotoxicity

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25–26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by 32P‐postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case‐by‐case basis, depending on the compounds profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance. Environ. Mol. Mutagen. 35:222–233, 2000

Hepatic epigenetic phenotype predetermines individual susceptibility to hepatic steatosis in mice fed a lipogenic methyl-deficient diet☆

BACKGROUND/AIMS The importance of epigenetic changes in etiology and pathogenesis of disease has been increasingly recognized. However, the role of epigenetic alterations in the genesis of hepatic steatosis and cause of individual susceptibilities to this pathological state are largely unknown. METHODS Male inbred C57BL/6J and DBA/2J mice were fed a lipogenic methyl-deficient diet (MDD) that causes liver injury similar to human non-alcoholic steatohepatitis (NASH) for 6, 12, or 18 weeks, and the status of global and repetitive elements cytosine methylation, histone modifications, and expression of proteins responsible for those epigenetic modifications in livers was determined. RESULTS The development of hepatic steatosis in inbred C57BL/6J and DBA/2J mice was accompanied by prominent epigenetic abnormalities. This was evidenced by pronounced loss of genomic and repetitive sequences cytosine methylation, especially at major and minor satellites, accompanied by increased levels of repeat-associated transcripts, aberrant histone modifications, and alterations in expression of the maintenance DNA methyltransferase 1 (DNMT1) and de novo DNMT3A proteins in the livers of both mouse strains. However, the DBA/2J mice, which were characterized by an initially lower degree of methylation of repetitive elements and lower extent of histone H3 lysine 9 (H3K9) and H3 lysine 27 (H3K27) trimethylation in the normal livers, as compared to those in the C57BL/6J mice, developed more prominent NASH-specific pathomorphological changes. CONCLUSIONS These results mechanistically link epigenetic alterations to the pathogenesis of hepatic steatosis and strongly suggest that differences in the cellular epigenetic status may be a predetermining factor to individual susceptibilities to hepatic steatosis.