Engaging expert peers in the development of risk assessments.

The participation of external technical experts in the development of risk assessment documents and methodologies has expanded and evolved in recent years. Many government agencies and authoritative organizations have experts peer review important works to evaluate the scientific and technical defensibility and judge the strength of the assumptions and conclusions (OMB, 2004; IPCS, 2005; IARC, 2006; Health Canada, 2007; U.S.

Issues in the design and interpretation of chronic toxicity and carcinogenicity studies in rodents: approaches to dose selection.

For more than three decades chronic studies in rodents have been the benchmark for assessing the potential long-term toxicity, and particularly the carcinogenicity, of chemicals. With doses typically administered for about 2 years (18 months to lifetime), the rodent bioassay has been an integral component of testing protocols for food additives, pesticides, pharmaceuticals, industrial chemicals, and all manner of byproducts and environmental contaminants. Over time, the data from these studies have been used to address an increasing diversity of questions related to the assessment of human health risks, adding complexity to study design and interpretation.

Inhaled formaldehyde: exposure estimation, hazard characterization, and exposure-response analysis.

Formaldehyde has been assessed as a Priority Substance under the Canadian Environmental Protection Act. Probabilistic estimates of exposure of the general population in Canada to formaldehyde in ambient and indoor air are presented. Critical health effects include sensory irritation and the potential to induce tumors in the upper respiratory tract (the nasal region in rodents and potentially the lungs of humans).

Relevance of carcinogenicity bioassays in mice in assessing potential health risks associated with exposure to methylene chloride.

Considerable research has been conducted to identify possible mechanisms of the carcinogenicity of methylene chloride in rodents, and to ascertain whether the observed increased incidences of liver and lung tumours in mice exposed to this substance, are relevant in assessing the potential hazards and risks to human health. On the basis of a study that purported to show qualitative differences between murine and human tissues, in the subcellular localization of the Theta-class glutathione S-transferase enzyme responsible for converting methylene chloride to a putative highly unstable, but reactive genotoxic metabolite, it was suggested that the mouse is an inappropriate model for human health risk assessment. However, other studies conducted in vitro with intact cells do not support the hypothesis that a putatively reactive metabolite of methylene chloride must be generated only within the nucleus in order to be able to interact with genomic DNA.

Effects of benzothiophene on male rats following short-term oral exposure.

The systemic toxicity of benzothiophene, a sulfur-containing heterocyclic present in petroleum, coal, and their derived products, was studied in male rats following short-term oral exposure. Male Sprague-Dawley rats (130 +/- 20 g) (n = 5 per dose group) were treated with benzothiophene by gavage at dosages of 0, 2, 20 or 200 mg/kg/d for 21 d. In another study, male rats were treated with 0, 100, or 500 ppm benzothiophene via the diet for 28 d.

Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells.

Our aim was to identify the biochemical defect responsible for the inability of highly growth autonomous human tumor cells to proliferate in culture medium devoid of methionine, but containing homocysteine and 5-methyletrahydrofolic acid. We have adopted the terms "homocysteine-responsive" and "homocysteine-nonresponsive" to describe cells which can or cannot proliferate in methionine-free homocysteine-supplemented medium. Using a panel of genetically related homocysteine-responsive and -nonresponsive human melanoma cell lines, the results from a number of experiments indicate that acquisition of the "homocysteine-nonresponsive phenotype" is associated with the reduced intracellular accumulation of methyl-cobalamin, a critical cofactor of the methionine synthase enzyme.

Serum has a differential effect on DNA replication in a human melanoma cell line cultured in methionine or 5'-deoxy-5'-methylthioadenosine.

A human melanoma cell line called MeWo-LC1 exhibits a reduced ability to synthesize DNA when cultured in serum-supplemented medium containing 5'-deoxy-5'-methylthioadenosine (MeSAdo) in place of methionine. However, DNA replication in these cells occurs normally if the cells are cultured in serum-free medium containing transferrin, and MeSAdo in place of methionine. Although the presence of serum alters the cells' ability to respond to MeSAdo, it is not likely a consequence of any increased extracellular metabolism by MeSAdo-phosphorylase or adenosine deaminase activity, or due to the diminished uptake of the nucleoside.

Transformed rodent cells exhibit increased resistance to the carboxylic ionophores monensin and nigericin.

Rodent fibroblasts transformed with the Kirsten and Moloney murine sarcoma viruses exhibit increased resistance to the growth inhibitory and cytotoxic action of the carboxylic Na+/H+ ionophore, monensin. The inhibitory effect of monensin on cell proliferation requires exposure for periods longer than 24 hours. The virus-transformed cells also exhibit increased resistance to the K+/H+ ionophore, nigericin.

Reversion to a homocysteine-responsive phenotype in a human melanoma cell line is associated with diminished growth potential and increased methionine biosynthesis.

Our aim was to determine if the isolation of cells capable of proliferating in methionine-free homocysteine-containing medium, from the human MeWo-LC1 melanoma tumor cell line which is unable to proliferate or survive under such conditions, was associated with altered growth properties. Cells which were able to proliferate in methionine-free homocysteine-containing medium (homocysteine-responsive cells) were obtained from the homocysteine-nonresponsive MeWo-LC1 cell line after 8 months of continuous exposure to methionine-free homocysteine-containing medium. Unlike the parental MeWo-LC1 cell line, these homocysteine-responsive cells were also able to proliferate normally in methionine-free medium containing 5'-deoxy-5'-methylthioadenosine.

Agarose-selected variants of two human tumor cell lines exhibit altered methionine auxotrophy.

Our aim was to determine if the selection of human tumor cells with enhanced anchorage-independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK-N-MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose-selected variants were completely unable to proliferate in methionine-free medium containing its immediate precursor homocysteine.

Indomethacin shifts the peak of c-fos, egr-1, and c-myc gene expression in confluent fibroblasts induced by phorbol myristate acetate.

Phorbol 12-myristate 13-acetate (PMA) can induce transcription of a number of "early" genes in quiescent fibroblasts, although the biochemical steps intervening between activation of protein kinase c and changes in gene-regulatory proteins are only partially known. To investigate these pathways further, we have studied the effect of indomethacin (INDO) on the induction of expression by PMA of three early genes (c-fos, egr-1 and c-myc) in quiescent BALB/c 3T3 cells. INDO was found to markedly change the kinetics of PMA-induced c-fos and egr-1 gene expression, as measured by Northern analysis.

Altered methionine metabolism in metastatic variants of a human melanoma cell line.

In order to identify the biochemical defect(s) responsible for the reduced levels of DNA 5-methylcytosine (5-mCyt) found within highly metastatic (in athymic "nude" mice) variants of the poorly metastatic human melanoma cell line MeWo, the ability of these cells to grow in culture medium devoid of exogenous methionine but containing either homocysteine (Hcy) or 5'-deoxy-5'-methylthioadenosine (MeSAdo) was determined. In contrast to the parental MeWo tumor line, many (but not all) of these malignant variants were completely unable to proliferate in methionine-free homocysteine-containing medium. Many of these malignant variants also exhibited a reduced ability to proliferate in methionine-free MeSAdo-containing medium.

DNA methylating capacity in metastatic variants of a human melanoma cell line.

Certain highly metastatic (in athymic immunosuppressed "nude" mice) variants of the poorly metastatic human melanoma cell line MeWo have been found to contain dramatically reduced levels of DNA 5-methylcytosine compared to the parental cell line. To identify the underlying biochemical defect which could be responsible for the reduced DNA methylation within these cells, the intracellular ratio of S-adenosylmethionine/S-adenosylhomocysteine and level of extractable DNA-methyltransferase were examined. No significant difference in the ratio of S-adenosylmethionine/S-adenosylhomocysteine or extractable DNA-methyltransferase activity were found between the highly malignant variants and the parental cell line.

DNA (cytosine) methylation in murine and human tumor cell lines treated with S-adenosylhomocysteine hydrolase inhibitors.

The effects of periodate-oxidized adenosine, 3-deaza-adenosine and 3-deaza-(+/-)aristeromycin, potent inhibitors of S-adenosylhomocysteine hydrolase activity, on DNA methylation and the intracellular ratio of S-adenosylhomocysteine and S-adenosylmethionine have been examined in a series of murine and human tumor cell lines. A 24-h exposure of murine LC3, TA3 and B16 cells and human MeWo and K562 cells to 1-10 microM periodate-oxidized adenosine had a very slight inhibitory effect upon DNA methylation. 3-Deaza-(+/-)aristeromycin and 3-deaza-adenosine (50 microM) had virtually no effect upon DNA methylation in LC3 and B16 cells.

Reduced levels of DNA 5-methylcytosine in metastatic variants of the human melanoma cell line MeWo.

The total levels of DNA 5-methylcytosine were determined in a series of related highly metastatic cell lines which had been isolated from the poorly metastatic human melanoma tumor line MeWo. The procedure used to quantitate DNA 5-methylcytosine involved labeling of DNA with [6-3H]uridine, hydrolysis with formic acid, and separation of cytosine and 5-methylcytosine bases by high-performance liquid chromatography. The DNA 5-methylcytosine content of the parental MeWo tumor was 3.

Periodate-oxidized adenosine induction of murine thymidine kinase: role of DNA methylation in the generation of tumor cell heterogeneity.

We previously reported that thymidine kinase (TK) activity in a spontaneously TK-deficient (TK-) murine tumor cell line (called L61-M) could be partially restored following brief treatment of the cells in vitro with the potent DNA-hypomethylating agent 5-azacytidine. We now show here that similar results may be obtained by exposing cells in vitro to periodate-oxidized adenosine, a potent inactivator of the S-adenosylhomocysteine hydrolase enzyme. The ability of periodate-oxidized adenosine to induce TK activity within the L61-M cell line was dependent upon the concentration of drug used and the treatment period.

On the possible contribution of DNA hypomethylation to the induction of high frequency and heritable drug-induced alterations in the malignant phenotype.

A hypothesis is presented that anti-neoplastic agents can alter gene expression at very high frequencies, even in vivo, by altering DNA methylcytosine levels and/or patterns. Such changes can be inherited, either in a stable or unstable manner, and can lead to the generation of more, or less, malignant variants, depending upon the gene(s)-that is (are) affected. An experimental model is reviewed to support this hypothesis in which it is shown that thymidine kinase activity in (TK-) deficient tumor cell mutants can be switched on when exposed to the anti-neoplastic drug and potent demethylating agent, 5-azacytidine in vivo.

Induction of thymidine kinase activity in a spontaneously enzyme-deficient murine tumor cell line by exposure in vivo to the DNA-hypomethylating agent 5-aza-2'-deoxycytidine: implications for mechanisms of tumor progression.

We previously reported that thymidine kinase (TK) activity in a spontaneously TK-deficient (TK-) mouse tumor cell line (called L61-M) could be partially restored by brief exposure of the cells in vitro to the DNA-hypomethylating agent 5-azacytidine. We now show that similar results can be obtained by exposing L61-M cells growing in mice to the DNA-hypomethylating agent 5-aza-2'-deoxycytidine. The frequency of TK+ cells within the TK- L61-M cell population was increased approximately 26,000-fold by the in vitro 5-azacytidine treatment.

Characterization of in vitro immunoselected variants from a highly metastatic murine tumor for alterations in malignant behavior in vivo.

A new Ly-6.2- antigen-loss variant (called L61 -M1) of the highly metastatic DBA/2 mouse (Ly-6.2+) MDAY-D2 tumor has been obtained by means of a monoclonal anti-Ly-6.

Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.

Highly immunogenic "tum-" (non-tumorigenic in normal syngeneic hosts) clonal variants can be selected from a variety of poorly immunogenic and highly tumorigenic mouse cell lines at very high frequencies (e.g., greater than 80%) after treatment in vitro with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

5-azacytidine induction of thymidine kinase in a spontaneously enzyme-deficient murine tumor line.

During the course of our studies on murine tumor cell metastases, one of our variant lines (called L61-M) was found to be unable to incorporate [methyl-3H]thymidine into DNA, due to a spontaneous deficiency in thymidine kinase (TK) activity. L61-M cells are unable to proliferate in HAT selection medium and are resistant to bromodeoxyuridine (BrdU). TK activity in L61-M cells is 4.

Possible epigenetic mechanisms of tumor progression: induction of high-frequency heritable but phenotypically unstable changes in the tumorigenic and metastatic properties of tumor cell populations by 5-azacytidine treatment.

Treatment of a variety of highly tumorigenic mouse lines in vitro with chemical mutagens, such as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), can result in extraordinarily high frequencies (sometimes in excess of 90%) of strongly immunogenic clones unable to grow progressively in normal syngeneic hosts. These clones will, however, grow in immunosuppressed hosts and gradually regain tumorigenic ability in normal mice if maintained in long-term (several months-1 year) culture, i.e.

The stimulation of rat liver microsomal CDP-diacylglycerol formation by guanosine triphosphate.

GTP has been found to markedly enhance the formation of CDP-diacylglycerol in rat liver microsomes. Neither GDP, GMP nor the nonhydrolyzable analogues of GTP increased the synthesis of the liponucleotide. The GTP stimulation of phosphatidate cytidylyltransferase activity is inhibited by EDTA and NaF.