Cell-mediated mutagenicity in Chinese hamster V79 cells of dibenzopyrenes and their bay-region fluorine-substituted derivatives.

The polycyclic aromatic hydrocarbons dibenzo(a,i)pyrene and dibenzo(a,h)pyrene, each of which possesses two bay regions, and their bay-region difluorinated derivatives were tested for mutagenicity for ouabain and 6-thioguanine resistance in Chinese hamster V79 cells. Since V79 cells do not metabolize polycyclic aromatic hydrocarbons, mutagenesis was tested in both the presence and the absence of golden hamster embryo cells capable of metabolizing polycyclic aromatic hydrocarbons. Neither of the dibenzopyrenes nor their fluorinated derivatives were mutagenic in the absence of the golden hamster embryo cells. In the presence of these cells (cell-mediated assay), both dibenzopyrenes were mutagenic, whereas the difluorinated derivatives, 2,10-difluorodibenzo-(a,i)pyrene and 3,10-difluorodibenzo(a,h)pyrene, either were inactive or exhibited (on a dose basis) a weak response. However, the mutagenicity of the dibenzopyrenes was eliminated when they were coincubated with 7,8-benzoflavone, a mixed-function oxidase inhibitor. The results suggest that metabolic oxidation of these polycyclic aromatic hydrocarbons at the bay region (presumably to diol-epoxides) is required for a mutagenic response in the cell-mediated assay.

Characterization of mutations that feminize gametophytes of the fern Ceratopteris.

Gametophytes of the fern Ceratopteris are either male or hermaphroditic. Their sex is epigenetically determined by the pheromone antheridiogen, which is secreted by the hermaphrodite and induces male and represses female development in other young, sexually undetermined gametophytes. To understand how antheridiogen represses the development of female traits at the genetic level, 16 new mutations that feminize the gametophyte in the presence of antheridiogen were identified and characterized. Seven are very tightly linked to the FEM1 locus previously described. Nine others define another locus named NOTCHLESS1 (NOT1), as several of the not1 mutants lack a meristem notch. Some not1 mutations also affect sporophyte development only when homozygous, indicating that the not1 mutations are recessive and that NOT1 is also required for normal sporophyte development. The epistatic interactions among FEM1, NOT1, and other sex-determining genes are described. This information was used to expand the genetic model of the sex-determining pathway in Ceratopteris. On the basis of this model, we can say that the presence of antheridiogen leads to the activation of the FEM1 gene, which not only promotes the differentiation of male traits, but also represses female development by activating the NOT1 gene. NOT1 represses the TRA genes necessary for the development of female traits in the gametophyte.

Generating autotetraploid sporophytes and their use in analyzing mutations affecting gametophyte development in the fern Ceratopteris.

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.

Education, socioeconomic and lifestyle factors, and risk of coronary heart disease: the PRIME Study.

BACKGROUND: Socioeconomic differentials have been described in the risk of coronary heart disease (CHD) but the extent to which these differentials are explained by lifestyle factors has been examined to a lesser degree. We have examined the contribution of socio-economic factors to risk of CHD in a large cohort study in France and Northern Ireland. METHODS: In all, 10 593 men aged 50-59 years were examined between 1991 and 1994 in centres in Northern Ireland, Lille, Strasbourg, and Toulouse. Details were obtained for a number of socio-economic indicators from the men at the baseline examination. Men were also screened for evidence of CHD and followed annually by questionnaire for incident cases of coronary disease. Coronary events (coronary deaths, myocardial infarction, and angina) were documented by clinical records and were reviewed by an independent medical committee. RESULTS: In all, 842 men (8%) showed some evidence of CHD at screening examination and these men were more likely to be living in poorer material circumstances, be unemployed, or have had less full-time education than men without CHD at screening in both France and Northern Ireland. These relationships persisted following adjustment for all known risk factors for CHD. Among men who were initially free of CHD there were clear socio-economic differentials (years of full-time education, unemployment, and educational level) in the distribution of several risk factors for CHD, notably smoking habit (which differs in France and Northern Ireland), systolic blood pressure, body mass index, and fibrinogen. Total cholesterol in contrast showed no socio-economic differential whilst those with a shorter period of full-time education and the unemployed tended to be high consumers of alcohol. In this cohort of men free of CHD at baseline few socio-economic indicators showed relationships with risk of CHD by 5 years of follow-up. Only years in full education, educational level, and unemployment status when adjusted only for age and country showed significant relationships with CHD risk, but these became non-significant following adjustment for major CHD risk factors. CONCLUSIONS: Socio-economic differentials in long-term risk of CHD are apparent in both cohorts of men from France and Northern Ireland, particularly in men with evidence of CHD at baseline. Among men free of CHD at baseline, although there is strong evidence of socio-economic differentials in cardiovascular risk factors these do not contribute independently to risk of CHD at 5 years of follow-up in this large cohort of men from France and Northern Ireland.

Cadmium-induced 8-hydroxydeoxyguanosine formation, DNA strand breaks and antioxidant enzyme activities in lymphoblastoid cells.

The effect of cadmium ion (Cd) and ascorbic acid (Asc) on the induction of oxidative DNA damage and on the activities of antioxidant enzymes were investigated in human lymphoblastoid cells (AHH-1 TK+/-). Cd at low concentrations of 5-35 microM induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and caused nuclear DNA strand breaks. The formation both of 8-OHdG and of DNA strand breaks was dose-dependent at the low Cd concentration; both parameters were linearly correlated with each other (R = 0.932 and P = 0.0209). 8-OHdG formation by Cd plateaued at a Cd concentration of 50 microM. Asc also induced 8-OHdG formation, but it had no synergistic effect with Cd on the formation of 8-OHdG or DNA strand breaks. Cd at the concentration of 50 microM induced the nuclear activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD). Furthermore, Cd caused a decrease in the concentration of reduced glutathione (GSH) and an increase in concentration of the oxidized form (GSSG). While Asc had no observable effect on SOD activity, it did increase nuclear catalase activity in cells. This effect on catalase was synergistic with that of Cd. The linear correlation between 8-OHdG and DNA strand breaks induced by Cd at the lower Cd concentrations (< or = 50 microM), suggested that the extent of formation of DNA strand breaks induced by Cd may be offset by their induction of the formation of 8-OHdG and antioxidant enzyme activities.

Growth curves and survival characteristics of the animals used in the Biomarkers of Aging Program.

The collaborative Interagency Agreement between the National Center for Toxicological Research (NCTR) and the National Institute on Aging (NIA) was aimed at identifying and validating a panel of biomarkers of aging in rodents in order to rapidly test the efficacy and safety of interventions designed to slow aging. Another aim was to provide a basis for developing biomarkers of aging in humans, using the assumption that biomarkers that were useful across different genotypes and species were sensitive to fundamental processes that would extrapolate to humans. Caloric restriction (CR), the only intervention that consistently extends both mean and maximal life span in a variety of species, was used to provide a model with extended life span. C57BI/6NNia, DBA/2JNia, B6D2F1, and B6C3F1 mice and Brown Norway (BN/RijNia), Fischer (F344/NNia) and Fischer x Brown Norway hybrid (F344 x BN F1) rats were bred and maintained on study. NCTR generated data from over 60,000 individually housed animals of the seven different genotypes and both sexes, approximately half ad libitum (AL) fed, the remainder CR. Approximately half the animals were shipped to offsite NIA investigators internationally, with the majority of the remainder maintained at NCTR until they died. The collaboration supplied a choice of healthy, long-lived rodent models to investigators, while allowing for the development of some of the most definitive information on life span, food consumption, and growth characteristics in these genotypes under diverse feeding paradigms.

The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands.

We have utilized a validated (standardized) estrogen receptor (ER) competitive-binding assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding assay. Initially, test chemicals were screened at high concentrations to determine whether a chemical competed with [3H]-estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed using a wide range of concentrations to characterize the binding curve and to determine each chemical's IC50 and relative binding affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 x 10(6)-fold range of RBAs from several different chemical or use categories, including steroidal estrogens, synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens), phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the 100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4-dihydroxybenzophenone, and 2,2'-methylenebis(4-chlorophenol), compounds that have not been shown previously to bind the ER. It was also evident that certain structural features, such as an overall ring structure, were important for ER binding. The current study provides the most structurally diverse ER RBA data set with the widest range of RBA values published to date.

Role of nitroreduction in the synergistic mutational response induced by mixtures of 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium.

Previous studies showed that binary mixtures of the environmental pollutants 1- and 3-nitrobenzo[a]pyrene produced a synergistic mutational response in the Salmonella reversion assay. Since nitroreduction is believed to mediate the direct-acting mutagenicity of the individual compounds, we have examined the role of nitroreduction in the mutagenicity of mixtures of 1- and 3-nitrobenzo[a]pyrene in the Salmonella plate incorporation assay. While mixtures of 1- and 3-nitrobenzo[a]pyrene induced up to 183% more revertants in strain TA98 than produced by equivalent amounts of the individual compounds, in the nitroreductase-deficient strain TA98NR the same mixtures only induced up to 57% more revertants than the individual compounds. Analysis of mixtures of 1- and 3-nitrosobenzo[a]pyrene (the two-electron reduction products of 1- and 3-nitrobenzo[a]pyrene) for mutation induction in TA98 yielded no evidence of a synergistic effect between the compounds. The mutagenicity of the mixtures was dependent upon the amount of the more mutagenic component. Salmonella cultures were also incubated with mixtures of 1- and 3-nitrobenzo[a]pyrene, as well as with equivalent amounts of the individual compounds. In two experiments, nitroreductive ability, as measured by the amount of 1-nitropyrene metabolized to 1-aminopyrene in 1 hr, was increased 9 to 105% in cultures pretreated with the mixtures as compared with cultures pretreated with the individual compounds. The results of this study support the hypothesis that nitroreduction is a major factor in the synergistic mutational response induced by 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium.

The effects of unsubstituted polycyclic aromatic hydrocarbons on the growth of Escherichia coli.

The effect on growth of E. coli of a series of 3-, 4-, and 5-ring unsubstituted polycyclic aromatic hydrocarbons (PAH) is determined at concentrations of 10-5M, 10-6M, and 10-7M. The angular hydrocarbon configurations of 1,2-benzanthracene, 1,2,5,6-dibenzanthracene (DIBA), and 3,4-benzpyrene (BP) promote growth; tetracene and pyrene have little or no effect on growth while at most concentrations anthracene, phenanthrene chrysene, 1,2,3,4-dibenzanthracene, and pentacene inhibit bacterial growth. No universal pattern of concentration effect is manifest, but based on the results an angular acene configuration is a necessary condition for growth stimulation.

Reduced levels of catalase activity potentiate MPP+-induced toxicity: comparison between MN9D cells and CHO cells.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to be toxic by inducing oxygen free radicals in the mammalian nervous system, especially in the nigrostriatal dopaminergic system. The present study was designed to compare the toxic effects of MPP+, the active metabolite of MPTP, in MN9D neuronal cells that exhibit relatively low levels of catalase activity, as compared to CHO cells, which exhibit high levels of catalase activity. The survival of the MN9D cells in the presence of 250 microM MPP+ was less than 10%, whereas CHO cells exhibited 70% survival at the same concentration of MPP+. The ED50 values of MPP+ in MN9D and CHO cell lines were 60-600 microM, respectively. MN9D cells contain less catalase, an enzyme believed to be involved in the detoxification of free radicals compared to CHO cells. The catalase activity was 2 Units/mg protein in MN9D cells and 30 U/mg protein in CHO cells. The catalase activity in CHO cells increased with increasing MPP+ concentrations from 100-500 microM, however, it decreased at 1 mM MPP+. In contrast, catalase activity in MN9D remained the same at all MPP+ concentrations. When the CHO cells were pre-treated with 10-25 mM 3-aminotriazole (3-AT), which inhibits catalase activity, and exposed to MPP+ at various concentrations, they became susceptible to MPP+. It is evident from these data that the differential susceptibility to MPP+ in these two cell lines are due to differences in catalase activity. In addition, the inhibition of constituentive catalase activity in CHO cells by 3-AT treatment enhances their susceptibility. In conclusion, the study demonstrates that catalase activity represents an important defence mechanism in MPTP-induced toxicity.

Photodynamic effects of dyes on bacteria. III. Mutagenesis by acridine orange and 500-nm monochromatic light in strains of Escherichia coli that differ in repair capability.

In the presence of acridine orange (AO) and monochromatic 500-nm light, the recombination-deficient strain of Escherichia coli, WP10 (recA), showed a 15-fold increase in mutation rate over the wild-type (WP2) strain. Under the same conditions, strain Bs--1 (uvrB lexA lon) showed a 5-fold increase in mutation rate over strain WP2. In contrast, the endonuclease-deficient, strain, WP2s (uvrA), showed a lower AO-500 nm mutation rate than wild-type. The extremely high mutation rate of the recA strain cannot be due to error-prone inducible SOS repair since the inducible recA + function is absent. Repair of the AO-500 nm-induced lesions is likely due to a recA+-dependent, error-free, recombination process. It is concluded that the high mutation rates with AO-500 nm light obtained in chemostat cultures of recA and lexA strains occur as a consequence of errors during semi-conservative DNA replication in the presence of unrepaired DNA lesions.

Biological effects of dyes on bacteria. VI. Mutation induction by acridine orange and methylene blue in the dark with special reference to Escherichia coli WP6 (polA1).

Acridine orange (AO) and methylene blue (MB) in the dark were shown to be weak to moderate mutagens (induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. However, strain WP2 (wild-type) was not mutated by AO in the dark, in confirmation of earlier data. The presence of 2 microM AO reduced by 41% the spontaneous mutation rate in strain WP2, from 4.1 to 2.4 mutants/10(8) cells/generation. In the polymerase I-deficient strain WP6 (polA1), 2 microM AO increased the mutation rate in the dark 14-fold. We propose that both spontaneous and AO-induced mutagenesis in the absence of light occur at the site of semiconservative DNA replication. If the intercalation mechanism for the effects in the absence of light is valid, the wild-type strain (WP2) may be resistant to frameshift mutagenesis induced by intercalated compounds, while the polymerase I-deficient strain (WP6) may be highly suceptible to the presence of an intercalated dye such as AO at the DNA-replication fork. MB and AO likely act through different mechanisms since MB is only a moderate mutagen in strain WP6 and the other repair-deficient strains tested.

Photodynamic effects of dyes on bacteria. V. Mutagenesis by acridine orange and 460-nm or 500-nm light in strains of Escherichia coli that differ in repair capability.

With acridine orange (AO) and monochromatic 460-nm light, the mutation rate of the wild-type strain of Escherichia coli (WP2) was 3-fold higher than that of the endonuclease-deficient strain WP2 (uvrA). In addition, the mutation rates of the recombination-deficient strains WP10 (recA) and Bs-1 (uvrB lexA) were also about 3-fold less than that of wild-type strain. This observation is in striking contrast to the earlier results with AO and 500-nm light in which strains WP10 and Bs-1 yielded mutation rates that were 12-fold and 5-fold greater, respectively, than the wild-type response. The relatively large decrease in mutation rate when the uvrA endonuclease was absent together with structural considerations in the binding of AO to DNA lead us to propose that the major lesions leading to mutations produced by 460-nm light in the presence of AO may be true DNa single-strand breaks and occur before DNA replication.

Photodynamic effects of dyes on bacteria. II. Genetic effects of broad-spectrum visible light in the presence of acridine dyes and methylene blue in chemostat cultures of Escherichia coli.

Photodynamic mutagenesis was studied in chemostat cultures of Escherichia coli B/r (TlR trp) exposed to one of six different acridine dyes or methylene blue. Mutation to phage T5 resistance was induced with a broad-spectrum fluorescent-light source. All of the agents tested were photomutagenic; acridine yellow was the most efficient sensitizer and quinacrine was the least efficient. Quinacrine also was moderately mutagenic in the dark, in contrast to the other agents tested, which were not significantly mutagenic in the dark at the low concentrations tested for photomutagenesis. The mutation rate with acridine orange was directly proportional to both fluence rate and dye concentration over the ranges tested. Photomutation rates with acridine orange, proflavine and methylene blue were independent of growth rate of the chemostat cultures. These results are consistent with photomutagenesis occurring as the result of photochemical damage to DNA-dye complexes, independent of cell expression was approximately 2.5 generations for each of the photomutagens tested. This short expression delay supports an earlier segregational model for expression of phage resistance. The following results suggest that photodynamic mutagenesis is due mainly to intercalated dye molecules: (1) both acridine and 9-aminoacridine are photodynamic mutagens; (2) acridine inhibits photomutagenesis with acridine orange; and (3) neither putrescine or spermine, which bind to DNA without intercalating, inhibited photomutagenesis by acridine orange or proflavine.

Effects of caloric restriction in animals on cellular function, oncogene expression, and DNA methylation in vitro.

While the life-extending and disease-modulating effects of caloric restriction (CR) are well documented in whole animal studies and in correlative experiments using cells taken from CR animals, very few studies have used cells in culture after their removal from the CR-fed animal. In using this in vivo-->in vitro approach we have attempted to examine the proposition that the effects of CR can be transferred to individual cells by analyzing the cellular functions of proliferation and transformation, the activation of oncogenes, and the methylation of DNA as a function only of diet. Pancreatic acinar cells excised from CR-fed Brown-Norway rats and placed in rich medium showed different responses compared to cells from ad libitum (AL)-fed controls. CR had the effect of slowing growth rate and protecting against spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation over 14 passages of cells in culture. At the molecular level, cells from the CR animals showed reduced c-Ha-ras oncogene expression and mutation as well as reduced mutation of the p53 suppressor gene. CR also increased genomic methylation of ras DNA. We conclude that the effects of CR treatment of the animal are transferred to individual cells and note that these responses (decreased proliferation and transformation; depressed oncogene expression and mutation and decreased suppressor gene mutation; and increased oncogene methylation) are cellular and molecular analogs of in vivo weight loss, life extension, and carcinogenesis modulation, which are hallmarks of CR in the whole animal. The fact that these responses are seen generations after the cells are removed from the CR-treated animal indicates that CR causes a permanent predisposition of pancreatic acinar cells to these modulated responses and shows the value of the in vivo-->in vitro protocol in studies that relate diet to cellular and molecular function.

Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrenes in Salmonella typhimurium strains.

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.

Damage to DNA by cadmium or nickel in the presence of ascorbate.

The interactive effects of the anti-oxidant ascorbate (Asc) and the metals cadmium (Cd, as CdCl2) or nickel (Ni, as NiCl2) on the in vitro formation of breaks in double-stranded deoxyribonucleic acid (d/s DNA) were determined. Concentrations of 50 microM Cd or 200 microM Ni were dosed for 4 hours in factorial combinations with 500 microM Asc in RPMI 1640 medium (7 percent bovine serum) in which AHH-1 TK+/- cells (a spontaneously transformed human B lymphoblastoid cell line by Gentest Corp.) were replicating. In combination with Asc, Cd caused significant d/s DNA breaks (p < 0.01, n = 5), while Cd in the absence of Asc produced only a slight (but not significantly different) amount of d/s DNA damage when compared to the cells with no Cd added. The Asc alone was not damaging. The Cd caused damage to the d/s DNA only when Asc was present. The percent of d/s DNA remaining following the respective treatments was: +Cd+Asc, 13 +/- 3; +Cd-Asc, 46 +/- 8; -Cd+Asc, 54 +/- 5; -Cd-Asc, 55 +/- 7. Conversely, the presence of Ni resulted in increased amounts (percent) of d/s DNA compared to control values: +Ni+Asc, 63 +/- 5; +Ni-Asc, 58 +/- 5; -Ni+Asc, 52 +/- 1; -Ni-Asc, 51 +/- 4, (p < 0.05, n = 3). The contrasting results between Cd and Ni in the presence of Asc may reside in the point of action; while Cd acts directly on DNA, Ni is reported to act on heterochromatin. Although Asc is a recognized anti-oxidant, its presence in the media mixture potentiated d/s DNA damage from the Cd. This may be caused by a Fenton-type reaction in which an antioxidant in the presence of metal generates hydroxyl radicals and consequently d/s DNA breaks. Oxidative reactions between metals, oxygen, and antioxidants such as Asc may represent an important mechanism of cell death, toxicity, and transformation.

Actions and interactions of nickel and magnesium on the transformation response of transformed cells in culture.

Nickel (Ni) and magnesium (Mg) exert separate and interacting effects on cells: Ni is toxic while Mg enhances the transformation response of transformed cells and protects from heavy metal-induced toxicity. Transformed rat liver epithelial cells were used in the soft agar (SA) assay to measure the effect of Ni and/or Mg on the expression of anchorage independence. Cells were exposed to +/- Ni and +/- Mg in a single passage of growth medium (GM) prior to assay in SA. The cells were then treated with +/- Ni and +/- Mg in the SA resulting in a 4 x 4 treatment matrix yielding 16 Ni/Mg combinations. Nickel was expected to decrease the transformation frequency (TF) and did so in 6 of the 16 cases. Magnesium was expected to enhance the TF independently of Ni; Mg increased TF values in 7 of 16 cases. The Ni-Mg interaction occurred in 11 of 16 cases. In general, Mg and Ni effects were observed more in GM than is SA. It is not evident from this study why the Ni, Mg, and Ni-Mg effects are not observed universally, but it is evident that metal-metal interactions are not simply defined or analyzed in biological systems. A refined factorial design may be useful in further separating such interactions. From the point of view of the implementation of the SA assay, in which test substances are typically dose previous to the implementation of putting the exposed cells in SA, it is clear that assay results can be markedly altered by the presence of the test compound in the soft agar.

Hyaluronic acid-filled mammary implants: an experimental study.

Issues of radiolucency and biocompatibility of currently available mammary implants have prompted the search for alternatives. Several new filler materials have been suggested recently but have involved the use of materials foreign to the body. We have studied the use of a naturally found polysaccharide molecule, hyaluronic acid, as an alternative filler material to silicone gel. We tested hyaluronic acid-filled implants using standard mammographic techniques, applanation tonometry, and in an in vivo animal model (n = 24) up to 1 year after implantation. The present study demonstrates that hyaluronic acid-filled implants have softness comparable with that of silicone gel and saline implants and are more radiolucent, allowing better visualization of breast structures around the implant. Furthermore, in vivo studies fail to demonstrate any adverse reactions to the material over a period of 1 year. Hyaluronic acid has unique properties in modulating the process of wound healing, and these properties may be applied to the tissues surrounding the implants as a result of leaching of hyaluronic acid through the covering shell. Although further studies using larger volumes of filler, characterization of the hyaluronic acid within the implant, quantification of the exact amounts of hyaluronic acid leached into surrounding tissues, and a more appropriate primate model need to be undertaken, this pilot study points out that there may be more biologically compatible materials for the use in breast implants that warrant further investigation.

Does caloric restriction induce hormesis?

The question of whether caloric restriction (CR) is hormetic is addressed in terms of two common definitions of the term. In terms of the older definition, i.e., a growth-stimulatory effect when lower doses of a compound which resulted in growth inhibition at higher doses, CR is better characterized as a co-hormetic (i.e., a paradigm which at relatively "low doses," in combination with some stimulus, will evince increased growth (proliferation) and at higher "doses" will inhibit this increased proliferation) rather than a hormetic agent. Mechanisms such as cellular selection of cellular subpopulations, increases in receptor efficiency, and preservation of cellular proliferative potential can interact with agents and produce increased growth as long as the CR is not too severe. In terms of a broader definition, i.e., nonmonotonic dose-response behavior of a compound for any adverse response, CR appears to be hormetic, both as a result of body weight (BW) loss and other potential mechanisms. The impact of changes in BW, or frank CR, can be considered a component of every test for hormesis, and is thus capable for interaction with any other agent. The changes that BW loss (or CR) induce are so profound that any aspect of an agent's action - metabolism, pharmacokinetics, pharmacodynamics - can modulate the response of an organism to an agent. Similarly, other effects of a chemical that induce BW loss, e.g., physical activity or temperature dysregulation, can also induce dose-response curves that appear hormetic. The interaction of the hormetic agents of BW loss and CR can influence agent tests. Controlling these factors may make it possible to dissect the key components of a hormetic response. In addition, the effects of CR or BW loss appear to extrapolate well across species [Colman R, Kemnitz JW. Aging experiments using nonhuman primates. In: Yu BP (Ed), Methods in Aging Research. CRC Press, Boca Raton, FL, 1999, pp. 249-267]. Thus there is some reason to believe that these hormetic factors may be important for humans, and may already be a factor for tests of potentially adverse agents already conducted in humans.