PubMed Health⌕ Search

Biomedical subjects

K Earley

Publications and source records attributed to K Earley.

32 records · Page 2Linked to original sources
PubMed1991

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus).

1. The in vitro metabolism of [3H]benzo[a]pyrene (BP) and [14C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo. 2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of BP-4,5-diol. 3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides. 4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates. 5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25-30 days. The major adduct was anti-BPDE-deoxyguanosine.

Animals↗
PubMed1991

DNA adducts in rat lung, liver and peripheral blood lymphocytes produced by i.p. administration of benzo[a]pyrene metabolites and derivatives.

DNA adducts produced in vivo in rat lung, liver and peripheral blood lymphocytes following the i.p. administration of several synthetic benzo[a]pyrene (B[a]P) metabolites and ring-substituted derivatives have been analyzed by the nuclease P1 version of the 32P-postlabeling assay. These include 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11- and 12-hydroxy-B[a]P, (+/-)-B[a]P-trans-4,5-dihydrodiol, (+/-)-B[a]P-trans-7,8-dihydrodiol, (+/-)-B[a]P-trans-9,10-dihydrodiol and B[a]P-7,8-dione. Among the monohydroxy derivatives, only 2-, 9- and 12-hydroxy-B[a]P produced detectable adducts. The only disubstituted derivative studied that produced adducts was the trans-7,8-dihydrodiol. The resulting DNA adducts were compared to those produced in each tissue by administration of B[a]P. 9-Hydroxy-B[a]P and B[a]P-trans-7,8-dihydrodiol each lead to the formation of major B[a]P adducts seen in lung and liver respectively. None of the adducts derived from either 2-hydroxy-B[a]P or 12-hydroxy-B[a]P were observed following administration of B[a]P alone.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

Formation and persistence of novel benzo(a)pyrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA.

Male CD rats were injected with single i.p. doses of benzo(a)pyrene (B(a)P), and peripheral blood lymphocytes (PBLs), livers, and lungs were removed at various times after administration. DNA adducts were analyzed in each tissue by 32P postlabeling with nuclease P1 enhancement. Sister chromatid exchange frequencies were concomitantly measured in cultured whole blood. B(a)P-DNA adducts were observed in all three tissues from animals sacrificed between 1 and 56 days after injection. Maximal adduction levels occurred at about 4 days after administration, followed by a gradual loss of adducts over the period examined. The apparent half-lives of total DNA adducts were 15 days in liver, 17 days in PBLs, and 22 days in lung. Induced sister chromatid exchanges were linearly related to the amount of DNA adducts remaining in the PBLs at the time of harvest up to 56 days and were significantly elevated above concurrent controls up to 14 days. One of the major adducts found in each tissue was N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a) pyrene]yl)deoxyguanosine. An additional novel major adduct was found in the liver DNA and is derived from the further metabolism of B(a)P-trans-7,8-dihydrodiol. A second major novel B(a)P adduct was found in the DNA of lung tissues and accounts for about 40% of the total adducts present. Experimental evidence suggests that this adduct is derived from a metabolic pathway that includes the formation of 9-hydroxy-B(a)P.

Adenosine Triphosphate↗
PubMed1990

Formation and persistence of DNA adducts in the liver of brown bullheads exposed to benzo[a]pyrene.

The formation and persistence of benzo[a]pyrene (BP)-DNA adducts in the liver of brown bullheads (Ictalurus nebulosus) treated with the hydrocarbon (20 mg/kg body wt, i.p.) was investigated using the 32P-postlabeling assay. The highest level of covalent binding of BP to liver DNA (188 fmol BP adducts/mg DNA) was observed 25-30 days following treatment. After 70 days, the adduct level in liver DNA had declined to approximately 26% of the maximum adduct level. One major BP-DNA adduct and several minor ones were detected in the liver. The major adduct co-chromatographed with anti-BP-7,8-diol-9,10-epoxide-deoxyguanosine (anti-BPDE-dGuo) adduct. The data suggest that brown bullheads metabolically activate BP by the same mechanism as the mammalian systems susceptible to carcinogenic effects of the hydrocarbon.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

Formation and removal of DNA adducts in target and nontarget tissues of rats administered multiple doses of 2-acetylaminophenanthrene.

2-Acetylaminophenanthrene (2-AAP) is carcinogenic for the mammary gland, small intestine and ear duct in rats, but is not hepatocarcinogenic. In order to understand the tissue specificity of tumor induction, the formation and removal of DNA adducts in target and nontarget tissues have been compared in rats administered 2-AAP. Male and female Sprague--Dawley rats were treated i.p. with up to four weekly doses of 5 mg 2-AAP per kg body weight. 32P-Post-labeling analysis of the DNA from all tissues showed two predominant adducts (greater than 85-95% of the total binding) and at least four minor adducts. By comparing the results obtained from reacting N-hydroxy-2-aminophenanthrene with DNA at pH 5, the major adducts were identified as N-(deoxyadenosine-8-yl)-2-aminophenanthrene and 1-(deoxyguanosin-N2-yl)-2-aminophenanthrene. Two minor adducts, N-(deoxyadenosine-8-yl)-2-aminophenanthrene and 1-(deoxyadenosin-N6-yl)-2-aminophenanthrene, were also formed in the in vitro reactions. The distribution of adducts, extent of binding and adduct persistence were similar between target and nontarget tissues, which indicates that the tissue specificity of 2-AAP for tumor induction is due to factors in addition to adduct formation.

Animals↗
PubMed1989

Sensitivity of rat and mouse peripheral blood lymphocytes to BaP adduction and SCE formation.

Both mice and rats were injected i.p. with doses of benzo[a]pyrene (BaP) ranging from 10 to 100 mg/kg to compare species sensitivity to and the relationship between sister chromatid exchange (SCE) induction and DNA adduct formation. Twenty-four hours after injection, blood was removed by cardiac puncture and the peripheral blood lymphocytes (PBLs) were analyzed for both DNA adduct formation by 32P-postlabeling and SCE induction following lymphocyte culture. BaP induced similar, but not identical, SCE dose-response curves for each species. After BaP administration, the major DNA adduct, N2-[10 beta-(7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene)yl]deoxyguanosine (BPDEI-dGuo), was approximately 10-fold more prevalent in the PBLs of the mouse than those of the rat. Thus, for equivalent amounts of BPDEI-dGuo, a greater number of SCEs are induced in the rat than the mouse.

Animals↗
PubMed1988

Hepatic DNA adduct formation in rats treated with benzo[f]quinoline.

The formation of hepatic DNA adducts in male Sprague-Dawley rats following i.p. administration of benzo[f]quinoline (BfQ) was examined using a 32P-post-labeling assay. BfQ exhibited a low binding (11-27 amol adducts/microgram DNA) to liver DNA. Two BfQ-nucleoside adducts (one major and one minor) were detected. The BfQ-DNA adducts formed in vivo were chromatographically distinct from the adducts formed by the reaction of calf thymus DNA in vitro with BfQ-5,6-oxide, syn-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBfQ, anti-9 alpha,10 beta-dihydroxy-7 alpha,8 alpha-epoxy-7,8,9,10-tetrahydroBfQ, or anti-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydroBfQ-N- oxide. These results suggest that the bay-region diol epoxide of BfQ, unlike the bay-region diol epoxide derivatives of polynuclear aromatic hydrocarbons, is not involved in the covalent binding of BfQ to DNA.

Animals↗
PubMed1988

Analysis of DNA adducts in putative premalignant hepatic nodules and nontarget tissues of rats during 2-acetylaminofluorene carcinogenesis.

Exposure of rats to a standard four-cycle feeding regimen of 0.06% 2-acetylaminofluorene (AAF) results in the formation of putatively premalignant hepatic nodules, but the types and magnitude of DNA adducts formed in these nodules has not been previously examined. By using a sensitive 32P-adduct assay (R. C. Gupta, Cancer Res., 45: 5656-5662, 1985), we analyzed the DNA adduct lesions in individual hepatic nodules at various times during and after exposure to AAF. Kidney, spleen, and testis were included as nontarget tissues. No qualitative difference was observed in the DNA adducts found in hepatic nodules and nontarget tissues; however, quantitative differences occurred. At least one unknown and two known (dG-C8-AF and dG-N2-AAF) DNA adducts were detected, with dG-C8-AF being predominantly (96-98%) formed, in all tissues examined. At the end of the first three weeks of AAF feeding, the concentration of the deacetylated adduct dG-C8-AF in liver (223 fmol/micrograms DNA) was found to be about 2, 6, and 5 times higher than in kidney, spleen, and testis, respectively. The concentration of the N2-acetylated adduct in liver (4.5 fmol/micrograms DNA) was 4-fold higher than in kidney and strikingly higher (51- and 42-fold, respectively) than in spleen and testis. At the end of the fourth feeding cycle, total DNA adducts measured in the hepatic nodules ranged from 30-100 fmol/micrograms DNA, while the "surrounding liver," kidney, spleen, and testis showed 235, 218, 62, and 28 fmol adducts/micrograms DNA, respectively. Sixty days following the cessation of AAF, the binding in both the persistent nodules and liver had decreased to 7% of their respective levels measured at the end of the fourth cycle, while adducts in kidney, spleen, and testis were 32%, 18% and 19%. After 88 days, the binding levels in the nontarget tissues declined further, but no additional adduct removal occurred in the nodules. Our data indicate that (a) although the metabolic apparatus for activation of AAF is diminished in the hepatic nodules, a significant level of adduct formation occurs in the cells of this putative, premalignant lesion, and (b) unlike in the nontarget tissues, repair processes in the premalignant nodules may not be operative several weeks after the cessation of AAF exposure.

2-Acetylaminofluorene↗
PubMed1988

Use of human peripheral blood lymphocytes to measure DNA binding capacity of chemical carcinogens.

Although animal models have been used successfully to study metabolic activation and binding of carcinogens to DNA, only limited studies have been done in human systems. To circumvent the problems associated with the inaccessibility of human tissues and a lack of sensitive methods to detect DNA damage, we have investigated the capability of human peripheral blood lymphocytes in vitro to metabolize carcinogens to their DNA binding species by a 32P-labeled adduct assay. Freshly isolated lymphocytes were exposed at 37 degrees C for 18 hr to 4-aminobiphenyl, 2-aminofluorene, 2-anthramine, 2-acetylaminophenanthrene, benzidine, 1-nitropyrene, 1,2-benzanthracene, triphenylene, 7,12-dimethylbenz[a]anthracene, or benzo[a]pyrene at 30 microM each, compounds that are shown or suspected to be carcinogenic in experimental animals. Anthracene, pyrene, and perylene were included as noncarcinogenic controls. Our data indicate that all test carcinogens formed readily measurable levels of DNA adducts. Analysis of exposed DNAs by 32P-labeling after digestion and adduct enrichment showed exclusively or predominantly one major adduct for all test carcinogens, except for 2-anthramine, triphenylene, and 7,12-dimethylbenz[a]anthracene, which showed two or three adducts, in the range of 8-1500 amol/micrograms of DNA. No DNA binding was detected for the noncarcinogens. From 12 lymphocyte specimens studied thus far, significant interindividual variations were observed for 2-aminofluorene (62-fold), 7,12-dimethylbenz[a]anthracene (10-fold), benzidine (19-fold) and benzo[a]pyrene (18-fold) in their capacity to bind to the lymphocyte DNA. The lymphocyte system in combination with the 32P-adduct assay may prove to be an ultrasensitive means to determine interindividual variations in the ability to biotransform carcinogens.

Adenosine Triphosphate↗
PubMed1988

32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures.

A 32P-adduct assay for the measurement of low levels (1 adduct per 10(7) nucleotides) of binding of carcinogens to DNA has been reported previously. In this procedure, DNA is enzymatically hydrolyzed to 3'-monophosphates of normal nucleosides and adducts, which are 5'-32P-labeled by T4 polynucleotide kinase and [gamma-32P]ATP. Labeled adducts are resolved by TLC. Enrichment of adducts by extraction in 1-butanol [Gupta, R.C. (1985) Cancer Res., 45, 5656] or digestion with nuclease P1 [Reddy, M.V. and Randerath, K. (1986) Carcinogenesis, 7, 1543] prior to 32P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 10(9-10) nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. We have now compared recoveries for greater than 70, different carcinogen-DNA adducts of known and unknown chemical nature in the two enrichment procedures as well as in a new procedure in which polynucleotide kinase is substituted for nuclease P1. When compared with the butanol extraction procedure, arylamines (such as 2-aminofluorene, 2-aminophenanthrene, 2-naphthylamine, 4-aminobiphenyl, 4-azoaminobenzene and N'-acetylbenzidine) bound to the C8 position of guanine were lost almost completely (0.2-4% recovery) in the nuclease P1-mediated assay, but the presence of a polar group in the aromatic amine moiety (such as 2-acetylaminofluorene, 2-acetylamino-phenanthrene and methyl-4-azoaminophenyl) rendered similar recovery. In contrast, aromatic amines (2-amino-phenanthrene, 2-acetylaminophenanthrene, 2-acetylaminofluorene and methyl-4-azoaminobenzene) and polycyclic aromatic hydrocarbons (benzo[a]pyrene, bromomethylbenzanthracene and benzanthracene) bound to the exocyclic positions of guanine or adenine showed extensive or as complete recovery in the nuclease P1 procedure as in the extraction procedure. Some of the unknown presumably polar adducts showed a lower recovery (30-70%) in the butanol procedure as compared to the nuclease P1 enrichment. The recovery pattern of most adducts examined in the polynucleotide kinase-enrichment assay was essentially the same as found in nuclease P1-mediated assay, except that overall lower values were obtained. Our data suggest that a given DNA sample should be analyzed by different versions of the 32P-adduct assay, particularly, DNA of specimens of humans exposed to low levels of unknown carcinogens. The observation that chemical structure of an adduct may be detrimental in its recovery in the enzyme- and extraction-mediated enrichment procedures may serve as a probe in the structural characterization of adducts of unknown carcinogens.

Butanols↗
PubMed1987

32P-postlabeling analysis of liver DNA adducts in rats chronically fed a choline-devoid diet.

Liver DNA, obtained at various time intervals from rats chronically fed a choline-devoid diet, was analysed for the presence of aromatic or alkyl adducts by the 32P-postlabeling assay. Alkyl adducts were not detected. Aromatic DNA adduct lesions were revealed, but only at levels (1 adduct per 0.5-3 X 10(9) nucleotides) which are at the limits of the extremely high sensitivity of the method used, levels which remained constant throughout the period of feeding. Thus, contamination of the total environment of the animals with chemical carcinogens does not appear to be responsible for the genesis of the hepatocellular carcinomas that develop in rats chronically fed a choline-devoid diet. The diet, therefore, either acts as a complete carcinogen, or promotes the evolution to cancer of endogenous, 'spontaneously' initiated liver cells.

Animals↗
PubMed1985

32P-postlabeling analysis of peroxisome proliferator-DNA adduct formation in rat liver in vivo and hepatocytes in vitro.

Hepatocarcinogenic peroxisome proliferators, clofibrate, ciprofibrate, Wy-14643 or di(2-ethylhexyl)phthalate, were administered once daily by gavage to groups of three male F344 rats for 3 days and the rats were killed 2 h after the last dose. The DNA isolated from the livers was analyzed for possible carcinogen-DNA adducts, by a most sensitive 32P-postlabeling technique which can detect one adduct in 10(10) nucleotides. No adducts were detected by this assay in the DNA isolated from the livers of rats treated with any of the peroxisome proliferators. Adducts were also not found in the DNA of hepatocytes exposed in vitro to these peroxisome proliferators for 4 h in primary suspension cultures. Failure to detect peroxisome proliferator DNA adducts in hepatocytes under in vivo and in vitro conditions supports the contention that formation of a peroxisome proliferator-DNA adduct is not an essential step in the carcinogenesis by this novel class of carcinogens.

Animals↗
PubMedDate not supplied

Tumor-suppressive activity of CD66a in prostate cancer.

CD66a (human homolog of rat cell-cell adhesion molecule, also known as biliary glycoprotein) is a cell surface protein of the immunoglobulin family. CD66a has been shown to mediate homotypic cell adhesion. Aside from this, no other functions of this molecule have been demonstrated. We have observed previously that CD66a protein expression is lost in most prostate tumors, suggesting that the down-regulation of CD66a is associated with the abnormal growth of prostate cells. CD66a is homologous (65% identity) to rat cell-cell adhesion molecule, which has been shown to have tumor-suppressive activity. This homology suggests the possibility that CD66a might also be a tumor suppressor. In this report, we show that restoring CD66a expression in DU145 human prostate cancer cells by adenovirus (Ad)-mediated gene transfer dramatically altered the malignant phenotype of these cells, as evidenced by their reduced ability to form tumors in a xenograft animal model. This result suggests that loss of CD66a protein plays an important role in the development of prostate cancer, and that restoring CD66a expression might provide an effective treatment for prostate cancer. We further explored the possibility of using Ad vectors to deliver CD66a as a potential therapeutic agent for prostate cancer. Direct injection of Ad-CD66a, an Ad vector carrying the CD66a gene, into DU145 tumors in mice significantly suppressed the growth of these tumors. This antitumor activity of CD66a was found to be dose-dependent. These results suggest that CD66a has tumor-suppressive activity and that Ad-CD66a is a potential therapeutic agent for prostate cancer treatment.

Adenoviridae↗