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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. EPA, 2006). Expert advice has been solicited in other forms of peer involvement, including peer consultation in, for example, the U.S. EPA's Voluntary Children's Chemical Evaluation Program (VCCEP). This article discusses how the principles and practices of peer review can be extended to other types of peer involvement activities (i.e., peer input and peer consultation) to develop high-quality risk assessment work products. A comprehensive process for incorporating peer input, peer consultation, and peer review into risk assessment science is outlined. Four key principles for peer involvement-independence, inclusion of appropriate experts, transparency, and a robust scientific process-are discussed. Recent examples of peer involvement in the development of Health Canada's Priority Substances and Domestic Substance List (DSL) programs under the Canadian Environmental Protection Act (CEPA) serve to highlight the concepts.

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). The majority of the general population is exposed to airborne concentrations of formaldehyde less than those typically associated with sensory irritation (i.e., 0.1 mg/m3). Based primarily upon data derived from laboratory studies, the inhalation of formaldehyde under conditions that induce cytotoxicity and sustained regenerative proliferation within the respiratory tract is considered to present a carcinogenic hazard to humans. At airborne levels for which the prevalence of sensory irritation is minimal (i.e., 0.1 mg/m3), risks of respiratory-tract cancers for the general population estimated on the basis of a biologically motivated case-specific model are exceedingly low. This biologically motivated case-specific model incorporates two-stage clonal expansion and is supported by dosimetry calculations from computational fluid dynamics analyses of formaldehyde flux in various regions of the nose and single-path modeling for the lower respiratory tract. The degree of confidence in the underlying database and uncertainties in estimates of exposure and in characterization of hazard and dose response are delineated.

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. Moreover, investigations employing semi-quantitative methods of mRNA hybridization are not convincing in identifying the subcellular localization of active Theta class glutathione S-transferase, and do not support the hypothesis of the differential subcellular localization of this enzyme within the nucleus of mouse, but not human cells. There is therefore, insufficient evidence to support the view that qualitative differences between humans and mice in the subcellular distribution of Theta-class glutathione S-transferase, renders carcinogenicity studies conducted with mice irrelevant in human hazard identification and risk assessment.

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. When in vitro methionine synthase assays were performed in the presence of exogenously added methyl-cobalamin, specific methionine synthase activity in extracts obtained from homocysteine-responsive cells was only twofold greater than that observed with extracts prepared from homocysteine-nonresponsive cells. However, when exogenous methyl-cobalamin was omitted from the enzyme assays, methionine synthase activity in extracts derived from homocysteine-nonresponsive cells was dramatically reduced, compared with the small decrease observed with homocysteine-responsive cell extracts. Compared with their homocysteine-responsive counterparts, homocysteine-nonresponsive cells exhibited increased levels of cobalamin efflux and decreased intracellular accumulation of methyl-cobalamin. There was a clear relationship between the abilities of these related melanoma cell lines to proliferate in methionine-free homocysteine-supplemented medium, and the extent of cobalamin loss and capacity of exogenously added methyl-cobalamin to stimulate in vitro methionine synthase activity. These results indicate a link between alterations in the intracellular trafficking and/or metabolism of cobalamin and the increased growth autonomy of human melanoma cells.

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. In the presence of methionine, MeSAdo appears to have a stronger cytostatic effect in medium containing serum than in serum-free medium supplemented with transferrin. MeWo-LC1 cells contain MeSAdo-phosphorylase activity as measured both in vivo and in vitro. The diminished replication of DNA in medium containing serum and MeSAdo is likely not due to the inhibition of polyamine synthesis by the nucleoside. These results indicate that serum (factors) can have an important influence upon the ability of MeSAdo to act as a methio-source for cells cultured in the absence of methionine.

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. Since monensin is known to have significant effects upon the function and activity of the Golgi apparatus and the intracellular trafficking and processing of endocytosed as well as cell-derived materials, the results suggest that alterations in the activities of the organelles and pathways involved with intracellular protein trafficking and processing likely make an important contribution to the biological and cellular properties of transformed cells.

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. In vitro growth rate, methionine requirement, and capacity to form colonies on soft agarose of these homocysteine-responsive cells were reduced compared to those of the homocysteine-nonresponsive parental MeWo-LC1 cell line. Unlike MeWo-LC1, these homocysteine-responsive cells were able to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure of the MeWo-LC1 cell line to proliferate in methionine-free homocysteine-containing medium may be due to a deficiency in the synthesis of methionine from homocysteine and 5-methyltetrahydrofolic acid. These results indicate that acquisition of a homocysteine-responsive phenotype in homocysteine-nonresponsive malignant human tumor cells is associated with a reduction in the autonomous growth potential of such cells.

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). We herein demonstrate that the same result can be obtained with the poorly mutagenic cytidine analogue, 5-azacytidine, a strong DNA hypomethylating agent. 5-Azacytidine and EMS were equally and comparably effective, or ineffective, in inducing tum- variants from three different highly tumorigenic mouse cell lines. Like mutagen-induced tum- variants, those obtained after 5-azacytidine treatment generated usually strong cytolytic T lymphocyte (CTL) responses in vitro, and could grow in immunosuppressed (nude mouse) hosts. However, pretreatment of the tumor cell lines with 5-azacytidine did not cause significant increases in mutations at several independent drug-resistant gene loci, whereas EMS did. It is known that treatment of cells with 5-azacytidine can induce transcriptional activation of "silent" genes through a reduction of DNA 5-methylcytosine content, a process that can also be effected by mutagenic DNA alkylating agents such as EMS and MNNG. We therefore hypothesize that an "epigenetic" mechanism (DNA hypomethylation) leading to activation and expression of genes coding for potential tumor antigens is involved in the generation at high frequency of tum- variants after "mutagen" treatment. The implications of these findings to mechanisms of tumor progression and the generation of tumor heterogeneity are discussed.

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. The frequency of L61-M TK+ cells was increased from between 200- and 1000-fold depending upon the routes of tumor and drug inoculation following the in vivo administration of 5-aza-2'-deoxycytidine. 5-Aza-2'-deoxycytidine treatment increased the proportion of L61-M TK+ cells by the induction of TK activity and not as a result of the selection of any preexisting TK+ cells from within the L61-M tumor cell population. While 5-aza-2'-deoxycytidine treatment increased the frequency of L61-M TK+ cells within the tumor cell population, it had no effect upon the survival times of mice bearing the L61-M tumor line. These results indicate that some anticancer chemotherapeutic drugs may be able to promote the diversification of tumor cell populations in vivo through the activation of previously quiescent genes as a result of alterations in the methylation of DNA.

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. Inhibiting DNA synthesis completely prevented the effects of periodate-oxidized adenosine from being observed. Periodate-oxidized adenosine had no obvious mutagenic effect upon the L61-M cell line and had a slight but significant inhibitory effect upon the methylation of the cytosine nucleotides which were incorporated into DNA during the treatment period. These results suggest that during tumor development, alterations in the relative levels of S-adenosylhomocysteine and S-adenosylmethionine may lead to the inhibition of DNA methylation, resulting in the activation of previously quiescent genes, thereby promoting the phenotypic diversification of tumor cell populations as well as their progression from a relatively benign to a highly malignant state.

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 3.07 +/- 0.16%. Metastatic variants of MeWo which had been isolated using a combination of in vitro and in vivo selection procedures had DNA 5-methylcytosine levels between 2.45 +/- 0.15% and 2.75 +/- 0.09%. Metastatic variants which were isolated using a single step in vivo selection procedure exhibited DNA 5-methylcytosine levels of 1.92 +/- 0.02% to 2.68 +/- 0.09%. No consistent decreases in DNA 5-methylcytosine content were found in "artificial" metastases arising in athymic "nude" mice following the i.v. inoculation of MeWo or its cloned sublines. These observations provide further support for the hypothesis that alterations in the cytosine methylation of DNA may play an important role in the generation of tumor cell heterogeneity and the progression of tumors from relatively benign to highly malignant states.

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. The variants exhibited no significant change in their total DNA 5-methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine-free medium. These variants were unable to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S-adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S-adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5-methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.

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. Thus, stable alterations in these cellular parameters are not likely responsible for the reduction in DNA 5-methylcytosine content which appears to occur during "progression" of the MeWo tumor line from a relatively benign to highly malignant state.

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. As opposed to the normal peak of mRNA at 30 min and 30-60 min for c-fos and egr-1, respectively, the appearance of each peak in the presence of INDO was delayed by 30-60 min, although the amount of mRNA was approximately normal. By contrast, c-myc mRNA levels remained low for at least 3 hr. This effect on gene expression required concentrations of INDO (0.25-1 mM) which have previously been shown to inhibit cAMP-dependent protein kinases. The effect was reversible: after 75 min treatment with INDO followed by removal of the drug, c-fos was induced with normal kinetics by PMA. Cycloheximide superinduced the expression of c-fos, egr-1 and c-myc after PMA treatment; INDO inhibited this superinduction. Although its mode of action is not yet defined, the ability of INDO to perturb the kinetics of expression 3 PMA-inducible genes may make it a useful drug to study the signal transduction pathways involved in gene induction.

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 4.2% of that found in the wild-type parental MDAY-D2 cell line. Treatment of L61-M with 5-azacytidine, a known inducer of DNA hypomethylation, resulted in the expression of TK activity. These observations suggest that the TK deficiency in the L61-M cell line was due in part to an alteration in the methylation pattern of DNA, resulting in the diminished expression of the TK gene. These results demonstrate the ability of 5-azacytidine to induce TK activity in a spontaneously enzyme-deficient murine tumor cell line.

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-6.2 antibody in an in vitro immunoselection technique. Whereas L61 -M1 grew poorly when inoculated subcutaneously into the syngeneic host, it grew and metastasized in a similar way to the parental MDAY-D2 tumor when inoculated into immunosuppressed, athymic nude mice. L61 -M1 as well as another Ly-6.2- variant of the same MDAY-D2 tumor (called L61 ) which is poorly metastatic in the syngeneic host salvaged exogenous fucose into glycoproteins and glycolipids at rates 5.5 and 7.8 times that of the parental MDAY-D2 line. In contrast, the Ly-6.2- variants exhibited a 50-70% decrease in the incorporation of exogenous mannose into glycoproteins and glycolipids. L61 -M1 and L61 also exhibited alterations in the structures of the oligosaccharide moieties linked to the cell surface glycoproteins and/or glycolipids. Thus, the in vitro immunoselection technique can be used to obtain a panel of variants with stable phenotypic alterations in their growth and metastatic capacities. Such mutants may, like previously described lectin-resistant mutants, be useful in studying the contribution of cell surface glycoproteins and glycolipids to tumorigenicity and metastasis.

The properties of rat liver microsomal CDPethanolamine: 1,2-diacylglycerol ethanolamine-phosphotransferase (ethanolaminephosphotransferase; EC 2.7.8.1) are studied with respect to metal ion and substrate concentration. The enzyme requires magnesium (20 mM) or manganese (1 mM) ions for optimum activity. Manganese ions are better activators of ethanolaminephosphotransferase activity than are magnesium ions. Calcium 1 mM) inhibits the magnesium-activated ethanolaminephosphotransferase by 86% and the manganese-activated enzyme by 57%. The Km for CDPethanolamine is 2.4 and 0.7 x 10(-4) M, in the presence of magnesium or manganese ions, respectively. ATP plus pantetheine significantly inhibit the manganese-activated ethanolaminephosphotransferase, while the magnesium-activated enzyme is inhibited by ATP plus pantetheine and slightly stimulated by ATP plus CoA. In the presence of either metal ion, ATP itself inhibits enzyme activity, while CoA or pantetheine when added alone have no effect. Evidence is presented indicating that the inhibition of ethanolaminephosphotransferase activity by ATP plus CoA or ATP plus pantetheine is not due to the formation of acyl-CoA or acyl-S-pantetheine esters. At the present time, however, the true mechanism of inhibition is unknown. The results indicate that the cellular levels of ATP, CoA, pantetheine, magnesium, manganese, and calcium ions may all play a role in the regulation of phosphatidylethanolamine biosynthesis.

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. GTP enhances the activity of the enzyme in a concentration-, time-, and temperature-dependent manner and preincubation of rat liver microsomes with GTP produces a persistently activated phosphatidate cytidylyltransferase. GTP reduces the Km for phosphatidic acid, but has no effect on either the Km for CTP or the Vmax of the reaction. GTP, by stimulating the activity of the phosphatidate cytidylyltransferase, enhances the formation of phosphatidylinositol from CTP, phosphatidic acid, and inositol. Evidence is presented suggesting that the mechanism by which GTP stimulates the activity of the phosphatidate cytidylyltransferase involves a covalent modification of the enzyme itself or a protein intimately associated with the phosphatidate cytidylyltransferase.

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. Cell lines established from "artificial" metastases of MeWo or its cloned sublines, exhibited no consistent reduction in their ability to grow in methionine-free medium containing either Hcy or MeSAdo. These observations suggest that alterations in S-adenosylmethionine(AdoMet)-dependent transmethylation reactions may contribute to "progression" of the MeWo tumor from a relatively benign to a highly autonomous and malignant state.

GTP has been found to markedly enhance the formation of CDPdiacylglycerol in rat liver microsomes. The magnitude of the GTP stimulatory effect was influenced by the fatty acid composition of the phosphatidic acid substrate. Heat or proteolytic treatment of microsomal membranes prevented GTP from stimulating liponucleotide formation. Treatment of microsomes with low levels of detergent reduced the stimulatory action of the GTP. The increased synthesis of CDPdiacylglycerol obtained by the addition of GTP to the incubation medium is due to an increase in CTP:phosphatidate cytidylyltransferase activity, rather than to a reduction in the breakdown of either the substrates or product of the reaction. The results suggest that some other protein or enzyme mediates the effect of GTP upon CTP:phosphatidate cytidylyltransferase activity.

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. In LC3 cells, periodate-oxidized adenosine, 3-deaza-adenosine and 3-deaza-(+/-)aristeromycin reduced the intracellular ratio of S-adenosylmethionine/S-adenosylhomocysteine approximately 20-, 6- and 16-fold, respectively. In murine B16 melanoma cells, periodate-oxidised adenosine, 3-deaza-adenosine and 3-deaza-(+/-)aristeromycin reduced the intracellular ratio of S-adenosylmethionine/S-adenosylhomocysteine approximately 17-, 13- and 32-fold, respectively. These observations indicate that the cytosine methylation of DNA appears to be relatively insensitive to changes in the intracellular ratio of S-adenosylmethionine/S-adenosylhomocysteine and that such metabolic disturbances are not likely to be the major biochemical alteration responsible for the reduced level of DNA 5-methylcytosine found within transformed and malignant cells.