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Biomedical subjects

L Recio

Publications and source records attributed to L Recio.

At least 37 records · Page 2Linked to original sources
PubMed1998

The butadiene metabolite, 1,2:3,4-diepoxybutane, induces micronuclei but is only weakly mutagenic at lacI in the Big Blue Rat2 lacI transgenic cell line.

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites, 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). We investigated the mutagenicity and induction of micronuclei by DEB in vitro in Rat2 lambda/lacI transgenic fibroblasts (Big Blue Rat2 cells, Stratagene, LaJolla, CA). Assays for mutagenicity and micronuclei induction were carried out at concentrations of 0, 2, 5, or 10 microM DEB for 24 hours. Exposure of cells to these concentrations of DEB resulted in approximately 100, 50, and 10% survival, respectively, compared with media controls. In independent replicate experiments, no statistically significant increase in lacI mutant frequency was observed in Rat2 cells at any of the DEB exposure concentrations when compared to media or solvent controls. However, regression analyses indicated a trend toward increasing mutant frequency with increasing DEB exposure concentration. Experiments to examine the induction of micronuclei by DEB revealed a concentration-dependent increase in micronuclei in Rat2 cells following exposure to DEB. These results indicate that DEB induces micronuclei in the absence of detectable gene mutation at lacI in Big Blue Rat2 cells. The induction of micronuclei but only weak mutagenicity at the lacI transgene is likely due to the poor recovery of deletions using this lambda shuttle vector system, demonstrating the need to investigate multiple endpoints of genotoxicity when considering the mutational activity of a compound.

Animals↗
PubMed1997

Oncogene and tumor suppressor gene alterations in nasal tumors.

The development of nasal tumors in humans and rodents is likely mediated through the accumulation of genetic alterations in genes that regulate cell proliferation, cell death and differentiation (oncogenes and tumor suppressor genes). By examination of the relationship between genetic alterations that are known to occur in human cancers with those induced in rodent tumors with defined carcinogenic exposures, biologically relevant mechanistic linkages of molecular events leading to tumors in rodents and humans can be established. Molecular genetic studies on nasal squamous cell carcinomas (SCC) in rats thus far have indicated the presence of oncogenes unrelated to the ras oncogene family and that p53 mutation occurs at a high frequency among the nasal SCC examined. The finding of p53 mutations in rat nasal SCC and the high prevalence of p53 mutations among human SCC, indicates that a common molecular alteration is shared between rodent and human SCC.

Animals↗
PubMed1997

In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure.

Ethylene oxide (EO) is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue mice at 6-8 (n = 4/group) and 8-10 (n = 5/group) weeks of age were exposed to 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week). At necropsy, T-cells were isolated from thymus and/or spleen and cultured in the presence of concanavalin A, IL-2, and 6-thioguanine [Skopek, T.R., V.E. Walker, J.E. Cochrane et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 7866-7870]. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency (Mf) in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5 +/- 0.9 x 10(-6) (average Mf +/- SE) at 2 weeks post-exposure, compared with 2.3 +/- 0.8 x 10(-6) in thymus of control mice. Dose-related increases in hprt Mfs were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt Mfs was observed in splenic T-cells, with greater mutagenic efficiency (mutations per unit dose) found at higher concentrations than at lower concentrations of EO. Average induced Mfs (i.e. induced Mf = treatment Mf - background Mf) in splenic T-cells were 1.6, 4.6, and 11.9 x 10(-6) following exposures to 50, 100, or 200 ppm EO, respectively, while the average control Mf value was 2.2 +/- 0.3 x 10(-6). In aliquots of lymphocytes (both B- and T-cells) isolated from spleen for analysis of lacI mutations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI Mf and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutation Res., submitted]. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin.

Administration, Inhalation↗
PubMed1997

Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure.

In the present study, the lacI mutant frequency was determined in the tissues of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of 15 male transgenic lacI B6C3F1 mice were exposed to either 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week) and were sacrificed at 0, 2, or 8 weeks after the last EO exposure. The lacI transgene was recovered from lung, bone marrow, spleen, and germ cells for determination of the lacI mutant frequency. The tissues selected for analysis were tumor target site tissues in chronic bioassays (lung tumors and lymphomas) and germ cells. The lacI mutant frequency in lung was significantly increased at 8 weeks post exposure to 200 ppm EO (6.2 +/- 2.2 vs. 9.1 +/- 1.5. p = 0.046). In contrast, the lacI mutant frequency in spleen and bone marrow at 2 and 8 weeks was not significantly increased in mice exposed to 200 ppm EO. The lacI mutant frequencies in male germ cells for 200 ppm EO-exposed mice were not increased compared to air controls at 2 and 8 weeks post-exposure. In a spleen cell fraction two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI mutant frequency. The increased lacI mutant frequency in these animals was likely due to mutant siblings that contained background G:C --> A:T transitions at CpG sites. These results demonstrate that a 4-week inhalation exposure to EO is mutagenic in lung. However, EO did not increase the frequency of mutations recovered at the lacI transgene in other tissues examined under the conditions used in the present studies. Since the mutational spectrum for EO in other systems consists of an increased proportion of large deletions, the lack of a mutagenic response in the tissues examined is likely due to the lack of recovery of large deletions in lambda-based shuttle vector systems. These data indicate that a primary mechanism of EO-induced mutagenicity in vivo is likely through the induction of deletions, not specific point mutations.

Administration, Inhalation↗
PubMed1997

Analysis of hprt mutations occurring in human TK6 lymphoblastoid cells following exposure to 1,2,3,4-diepoxybutane.

1,3-Butadiene (BD) is a rodent carcinogen that is bioactivated to at least two genotoxic metabolites, 1,2-epoxybutene (EB) and 1,2,3,4-diepoxybutane (DEB). The mutational spectrum for DEB at hprt in human TK6 lymphoblasts (TK6 cells) was determined and compared with the mutational spectrum from spontaneous mutants. A DEB exposure of 4 microM for 24 h resulted in an average 5-fold increase in the hprt mutant frequency. Hprt mutants for molecular analysis were isolated from TK6 cells exposed to 4 microM DEB for 24 h (51 DEB-induced mutants) and from a set of spontaneous mutants (n = 43) isolated from the same TK6 stock cell cultures. Molecular analyses of hprt mutations were done by reverse transcription-polymerase chain reaction (RT-PCR) of hprt mRNA or exon-specific genomic PCR amplification of hprt followed by DNA sequencing of PCR products. There was an increased frequency of A:T-->T:A transversions among the DEB-induced mutants compared to spontaneous mutants (9/51; 18% DEB-induced compared to 2/43; 5% in spontaneous (one-way Fisher's exact test; P < or = 0.05). DEB-induced hprt mutants also had an increased frequency of genomic deletions affecting the 5' region of hprt (7/51; 14% DEB-induced compared to 1/43; 2% in spontaneous). Therefore, DEB is a mutagenic carcinogen that can induce genotoxicity by large deletions, rearrangements or single base substitution mutations.

Base Composition↗
PubMed1997

Characterization of hprt mutations following 1,2-epoxy-3-butene exposure of human TK6 cells.

1,3-Butadiene (BD) is an indirect-acting mutagen that is bioactivated in laboratory animals to at least two mutagenic metabolites, 1,2-expoxy-3-butene (EB) and 1,2,3,4-diepoxybutane (DEB). In the present study, the cytotoxicity, mutagenicity and mutational spectrum at hprt were determined after EB-exposure of human TK6 lymphoblastoid cells (TK6 cells). EB was cytotoxic at concentrations ranging from 200 to 1000 microM x 24 h; at 400 microM x 24 h, the cell survival relative to unexposed controls was approximately 10%. Exposure of TK6 cells to EB (400 microM x 24 h) resulted in a 5-9-fold increase in the hprt mutant frequency. Molecular characterization of EB-induced hprt mutants indicated that 78% of the mutations at hprt were single base substitutions. A significant (P < 0.05) increase in A:T-->T:A transversions was observed compared with spontaneous hprt mutants isolated during these studies. All of the A:T-->T:A transversions in EB-induced mutants occurred with the A in the non-transcribed strand. These data indicate that a primary mode of genotoxicity induced by EB in human TK6 cells is the induction of single base substitutions.

B-Lymphocytes↗
PubMed1996

Mutagenicity and mutational spectra of 1,3-butadiene in the bone marrow of B6C3F1 lacI transgenic mice.

We are using B6C3F1 lacI transgenic mice to investigate the in vivo mutagenicity and mutational spectra of 1,3-butadiene (BD). Male B6C3F1 lacI transgenic mice were exposed by inhalation to 0, 62.5, 625, or 1250 ppm BD for 4 weeks (6 h/day, 5 days/week). Tissues were collected 2 weeks after the final exposure for mutant frequency analysis. The lacI- mutant frequency in bone marrow from BD-exposed mice was increased 2- to 3.5-fold over air control mice. Analysis of the mutational spectrum of lacI mutants isolated from the bone marrow of lacI mice exposed to 625 ppm BD indicated a shift in the mutational spectrum at A:T base pairs relative to air controls: 2/41 point mutations occurred at A:T base pairs in air controls versus 6/24 in BD-exposed animals; 10/40 mutations occurred at A:T base pairs in lacI mice exposed to 1250 ppm BD. A-->T transversions were found only in BD-exposed animals. Although there was a reduction in the proportion of GC-->AT transitions at 5'-CpG-3' sites recovered from the bone marrow of BD-exposed mice (25%) relative to air controls (50%), there was no difference in the contribution of GC-->AT transitions at 5'-CpG-3' sites to the mutation frequency in BD-exposed mice compared to air controls. Since the bioactivation of BD by B6C3F1 mice produces at least two mutagenic metabolites epoxybutene (EB) and diepoxybutane (DEB), studies on the in vivo mutational spectra begin to provide the framework for the ultimate identification of the BD metabolite(s) responsible for the specific mutations observed.

Animals↗
PubMed1996

Assessment of 1,3-butadiene mutagenicity in the bone marrow of B6C3F1 lacI transgenic mice (Big Blue): a review of mutational spectrum and lacI mutant frequency after a 5-day 625 ppm 1,3-butadiene exposure.

1,3-Butadiene (BD) is a carcinogen that is bioactivated to at least two genotoxic metabolites. In the present article, we review briefly our previous studies on the in vivo mutagenicity and mutational spectra of BD in bone marrow and extend these studies to examine the effect of exposure time (5-days vs. 4-week exposure to 625 ppm BD used in previous studies) on the lacI mutant frequency in the bone marrow. Inhalation exposure to BD at 625 ppm and 1,250 ppm mutagenic in vivo, inducing an increase in the transgene mutant and mutation frequency in the bone marrow. Analysis of the mutational spectrum in BD-exposed and air control mice demonstrated that BD exposure induced an increased frequency of mutations at A:T base pairs. There was no difference in the lacI mutant frequency determined in the bone marrow between a short-term exposure to BD (5 days) and a longer-term exposure (4 weeks). These data taken together demonstrate that inhalation exposure to BD induces in vivo somatic cell mutation.

Administration, Inhalation↗
PubMed1995

Increased frequency of mutations at A:T base pairs in the bone marrow of B6C3F1 lacI transgenic mice exposed to 1,3-butadiene.

We have examined the spectra of mutations in a collection of 74 lacI mutants isolated from the bone marrow of B6C3F1 lacI transgenic mice exposed to 1,250 ppm 1,3-butadiene (BD). Of the 49 independent mutations analyzed in the present study, 30 of 49 (61%) were point mutations at G:C base pairs, and 10 of 49 (20%) were point mutations at A:T base pairs. The remaining mutations consisted of small deletions and insertions and a single tandem change. An analysis of variance (ANOVA) on the frequency of specific mutations observed in each animal in this mutagenicity experiment [present study; also Sisk SC et al. (1994): Carcinogenesis 15:471-477] indicated that the frequency of point mutations at A:T base pairs is significantly greater (P < 0.05) in BD-exposed mice than in the air controls. In addition, there was a decrease (P < 0.05; Fisher's exact test) in the frequency of G:C-->A:T transitions at 5'-CpG-3' sites in BD-exposed mice (27%) relative to air controls (51%). These data indicate that subchronic exposures to BD induces an increased frequency of in vivo mutation at A:T base pairs in the bone marrow of B6C3F1 lacI transgenic mice.

Adenine↗
PubMed1995

p53 status in spontaneous and dimethylnitrosamine-induced renal cell tumors from rats.

Rats carrying the Eker tumor-susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT-->CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non-p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer.

Adenoma↗
PubMed1995

Immunohistochemical localization of p53, PCNA, and TGF-alpha proteins in formaldehyde-induced rat nasal squamous cell carcinomas.

Mutation of the p53 tumor suppressor gene is a common event in many human cancers and has been specifically associated with invasive squamous cell carcinoma of the human skin and respiratory tract. Alterations in the p53 gene have also been identified in certain rodent tumors, including formaldehyde-induced nasal squamous cell carcinomas. Overexpression of transforming growth factor-alpha (TGF-alpha) is associated with carcinomas of the head and neck and respiratory tract in human patients and formaldehyde-induced rat nasal squamous cell carcinomas. Sections of rat noses containing tumors and other formaldehyde-induced lesions from rats exposed to 15 ppm formaldehyde vapor were examined using immunohistochemical techniques to detect and identify potential relationships between the presence and distribution of p53, proliferating cell nuclear antigen (PCNA), and TGF-alpha proteins. The five tumors that had p53 mutations were for mutant p53 protein by immunohistochemistry and three of six tumors with no detected p53 mutations were also immunoreactive for p53 protein. The presence, pattern, and distribution of p53 staining in tissue sections depended on the morphology of the lesion. PCNA immunoreactivity was strikingly similar in pattern and distribution to p53 immunoreactivity. The pattern and distribution of immunoreactivity for TGF-alpha did not directly correlate with the other markers. Mutation of the p53 tumor suppressor gene may be an important step in the progression of formaldehyde-induced nasal carcinogenesis in the rat. This study demonstrated that immunohistochemistry is a useful tool for the identification of sites within tumors that might have p53 mutations.

Animals↗
PubMed1995

The use of transgenic mice for studying mutagenicity induced by 1,3-butadiene.

The availability of in vivo transgenic mouse mutagenesis assays provides the opportunity to study the induction of mutations that reflects the integration of pharmacokinetics, biotransformation. DNA repair responses, and tissue susceptibilities. Transgenic mice containing in vivo marker genes for mutagenicity within shuttle vectors also permit the determination of the resulting mutational spectra in tissues of mice following exposure to mutagens and carcinogens. Exposure of B6C3F1 lacI transgenic mice to the rodent carcinogen 1,3-butadiene by inhalation results in an increase in the overall lacI mutant frequency in bone marrow with an increased frequency of point mutations at A:T base pairs. These studies demonstrate the utilization of transgenic mouse models for establishing linkages between exposure to carcinogens, internal tissue dose, and in vivo mutational events.

Animals↗
PubMed1995

Epidemiological and mechanistic data suggest that 1,3-butadiene will not be carcinogenic to humans at exposures likely to be encountered in the environment or workplace.

1,3-Butadiene (BD) is a carcinogen in both rats and mice with mice being substantially more sensitive than rats. It is not known if BD poses a carcinogenic risk for humans. Findings from exposure assessment studies indicate that potential industrial exposure to BD in monomer, polymer, and end-user industries is typically < 2 p.p.m. Epidemiologic studies of persons occupationally exposed to BD are inconclusive. In vitro metabolism of BD in rats, mice and human tissues indicate that there are significant quantitative species differences in the metabolic activation of BD to butadiene monoepoxide (BMO) and butadiene diepoxide (BDE) and the detoxication of BMO. Activation/detoxication ratios calculated using in vitro kinetic constants reveal that ratios in mice were 12-fold greater than rats and humans. In rats and mice exposed to BD, concentrations of BMO in blood and tissues of mice were up to 14-fold higher than in rats and BDE was only detected in mice thereby providing a strong argument for why mice are highly sensitive to BD carcinogenicity. The fact that human tissues do not appear to metabolize BMO to BDE to any significant extent suggest that humans may not be sensitive to BD carcinogenicity. In mice, BDE is a more potent carcinogen than BMO. BDE is mutagenic in vitro at the hprt locus in human TK6 lymphoblasts at concentrations that were 100-fold less than the concentration of BMO required to yield a similar mutation frequency. Importantly, the concentrations of BDE that were genotoxic in vitro are nearly identical to the concentrations of BDE measured in blood and tissues of mice exposed to BD by inhalation. BD is genotoxic in mice, but not rats, following inhalation exposure and this is paralleled by species differences in observed tumor susceptibility. BD is not genotoxic in occupationally-exposed workers. The genetic basis for BD carcinogenicity appears to be primarily through induction of point mutations and deletion events mediated via the potent genotoxic metabolite, BDE. The genotoxic endpoints induced by BDE (e.g., deletion and point mutations) rather than BMO (e.g., point mutations) likely represent the underlying mechanism responsible for the striking species differences observed in the genotoxicity and carcinogenicity of BD in mice versus rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Characterization of cell lines derived from formaldehyde-induced nasal tumors in rats.

Cell lines derived from formaldehyde-induced nasal tumors in Fischer 344 rats were established. All of the lines were found to be epithelial and aneuploid and to express keratin, transforming growth factor-alpha, and epidermal growth factor receptor transcripts. Two of four lines were tumorigenic upon injection into nude mice, and these lines also contained point mutations in the p53 suppressor gene. The data indicate that these lines possess characteristics that make them a valuable tool for the study of chemically induced respiratory tract carcinogenesis.

Animals↗
PubMed1994

Transgenic animals in toxicology.

Recent advances have been made in the characterization of a number of transgenic animal models. These animal models have provided a powerful toxicological tool for studying in vivo chemical effects and have increased our understanding of the role of specific genetic alterations as predisposing factors for chemical carcinogenesis. The goal of this symposium was to introduce the development of transgenic animals and the utilization of transgenics in toxicology research focusing on understanding tissue-specific mutation, chemical effects, and cancer. The production of transgenic animals, including gene insertions and gene knockouts, and the utilization of transgenic technology for studying multistage carcinogenesis and tumor suppressor genes are described. Data on the application and implications of transgenics as a genetic endpoint are also discussed. The use of transgenic animals in toxicology should improve our understanding of the role of specific genetic alterations in the carcinogenic process and lead to improved estimations of human health risks.

Animals↗
PubMed1994

Absence of detectable P450 2E1 in bone marrow of B6C3F1 mice: relevance to butadiene-induced bone marrow toxicity.

The observation that the concentration of butadiene monoepoxide (BMO) is greater in bone marrow than in the blood following inhalation of 1,3-butadiene (BD) by B6C3F1 mice prompted the present investigation into whether cytochrome P450 2E1, the isozyme believed to be involved in the epoxidation of BD, is present in the bone marrow of B6C3F1 mice. The bone marrow of male and female B6C3F1 mice was analyzed by Western blot analysis and immunohistochemistry to determine whether cytochrome P450 2E1 is present, and, if present, in which cell types. Both Western blot and immunohistochemical analyses revealed the apparent absence of P450 2E1 in B6C3F1 mouse bone marrow. This observation suggests that BD is converted to BMO outside of bone marrow and is subsequently concentrated in the bone marrow, or that the conversion of BD to BMO occurs by an alternate enzymatic pathway within the bone marrow.

Animals↗
PubMed1994

Characterization of phosphodiester adducts produced by the reaction of cyanoethylene oxide with nucleotides.

Cyanoethylene oxide (CEO), a putative toxic and carcinogenic metabolite of acrylonitrile, is a direct-acting mutagen. The focus of this study was to elucidate potential adducts responsible for the mutagenic effect of CEO by characterizing products from the reaction of CEO with nucleotides. The reaction of CEO with the 5'-monophosphates of deoxyguanosine, deoxyadenosine, deoxycytidine or deoxythymidine resulted in the formation of at least one adduct for each nucleotide. Using two-dimensional NMR spectroscopy and fast atom bombardment mass spectrometry, CEO-nucleotide adducts (approximately 25% modification) were characterized as 2-cyano-2-hydroxyethyl phosphodiesters. The isolate from the reaction of deoxyguanosine-5'-monophosphate (dGMP) with CEO contained a second adduct, identified as N7-(2-cyano-2-hydroxyethyl)-dGMP. Single and double strand breaks, which were observed in supercoiled pBR322 plasmid DNA exposed to CEO (> 50 mM), may arise following formation of cyanohydroxyethyl phosphotriester adducts. The characterization of these phosphodiester adducts in vitro may provide insight into the intermediates responsible for the genotoxic effect of CEO in vivo.

Chromatography, High Pressure Liquid↗