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Biomedical subjects

A B Okey

Publications and source records attributed to A B Okey.

At least 73 records · Page 4Linked to original sources
PubMed1981

Rapid vertical tube rotor gradient assay for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the Ah receptor.

The Ah receptor, which regulates induction of aryl hydrocarbon hydroxylase (cytochrome P1-450), can be assayed using [3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. Sucrose density gradient (SDG) centrifugation is the most reliable method for detecting and quantitating Ah receptor; however, previous gradient assays in swinging bucket rotors (SBR) have required overnight centrifugation to achieve adequate separation of specific receptor peaks from nonspecific binding to other cytosolic components. These lengthy runs limit the number of assays which can be performed and could permit excessive dissociation of radioligand from the receptor. Thus, we tested brief (2-h) SDG centrifugation assays in a vertical tube rotor (VTR) as a possible alternative to prolonged SBR runs. Two-hour VTR runs yield gradient profiles very similar to those obtained with 16-h runs in an SBR and the calculated sedimentation coefficient (in low ionic strength buffer) is about 9.6 S for the Ah receptor assayed in either rotor. The concentration of Ah receptor in cytosols from liver, lung, kidney, and testis of C57BL/6J mice is the same by VTR analysis as when measured by SBR analysis. Thus, extensive dissociation of [3H]TCDD from the Ah receptor does not appear to occur in cytosols from a variety of tissues even with run times up to 16 h. Assays performed by rapid VTR centrifugation appear equally valid to those obtained by overnight SBR centrifugation.

Animals↗
PubMed1981

Aryl hydrocarbon hydroxylase activity in mouse, rat, and human mammary tumors.

Aryl hydrocarbon hydroxylase (AHH) activity was measured in microsomes from chemically induced and spontaneous mammary tumors of mice and rats and in 213 human breast tumors. Basal enzyme activities [pmol 3-hydroxybenzo(a)pyrene per mg protein per min] ranged from 0.05 to 0.5 for rat, 0.05 to 10 for mouse, and 0 to 40 for human tumors. For comparison, mean liver AHH activities were 13 in untreated rats and 100 in untreated mice. Thus, some human breast tumors had AHH activity exceeding that in rat liver. Injection of 80 mg beta-naphthoflavone per kg into tumor-bearing C3H/HeJ mice or Sprague-Dawley rats increased AHH activity to 10- to 70-fold over basal levels; there was no significant AHH induction in tumors from genetically "nonresponsive" DBA/2J or RF/J mice treated with beta-naphthoflavone, alpha-Naphthoflavone in the incubation flask inhibited AHH activity in some human breast tumors and stimulated activity in others, probably reflecting the presence of multiple forms of cytochrome(s) P-450 in the human tumor population. AHH activity in human tumors was not correlated with their estrogen receptor content. Since several drugs used in cancer treatment are substrates for polysubstrate monoxygenases, high levels of AHH activity in some human tumors may play a role in their response to chemotherapy.

Animals↗
PubMed1980

Ontogenetic expression of regulatory and structural gene products associated with the Ah locus. Comparison of rat, mouse, rabbit and Sigmoden hispedis.

The murine Ah complex represents a 'cluster' of genes controlling the induction of numerous cytochrome P-450-mediated monooxygenase 'activities' by polycyclic aromatic compounds. These forms of cytochrome represent structural gene products. A major regulatory gene product is a cytosolic receptor to which radiolabeled 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) binds with very high affinity. Developmental differences in mouse, rat and rabbit liver have been previously reported, indicating that some form of temporal control exists during the induction of enzymes associated with the ah locus. The cytosolic receptor levels were determined by sucrose density gradient centrifugation after dextran-charcoal treatment. Hepatic receptor levels, beta-naphthoflavone-inducible and control aryl hydrocarbon hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylase activities and total P-450 content were studied in the Sprague-Dawley rat, C57BL/6N mouse, New Zealand White rabbit and Sigmoden hispedis (cotton rat) as a function of age. 3-Methylcholanthrene-inducible and control aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activities in nonhepatic tissues of the neonatal and adult rabbit were examined. beta-Naphthoflavone-inducible hepatic microsomal proteins detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were also studied in the four species. Taken altogether, the developmental data indicate that: the form of P-450 responsible for induced aryl hydrocarbon hydroxylase activity is not the same as that for induced acetanilide 4-hydroxylase, and neither one is the same as induced cytochrome P-448; the presence of the TCDD-specific cytosolic receptor per se (detected as a distinct high-specificity saturable peak on sucrose density gradients) does not guarantee the expression of inducible aryl hydrocarbon hydroxylase or acetanilide 4-hydroxylase activity; although interesting developmental differences exist among all four species examined, the TCDD-specific receptor is maximal between the neonatal and weaning period, is considerably decreased in the adult, and is suppressed even more during the latter half of pregnancy; in general, the times at which the cytosolic receptor is highest or lowest parallels quite closely the well-known increases in inducible drug-metabolizing enzymes that have been commonly observed in the rat, mouse and other laboratory animals, and more studies are necessary before we understand what the 'TCDD-specific binding peak' (as observed on the sucrose density gradients) actually represents.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Pyrolysis products from amino acids and protein: highest mutagenicity requires cytochrome P1-450.

Pyrolysis products of proteins and amino acids are highly mutagenic, but metabolism of these chemicals by rat liver subcellular fractions is known to be required for production of the mutagenic intermediates. We examined the mutagenesis of seven purified pyrolysis products from tryptophan, lysine, glutamic acid, and soybean globulin with Salmonella typhimurium strain TA98 in the presence of liver fractions from genetically "responsive" C57BL/6N and Ah(b)/Ah(d) or "nonresponsive" DBA/2N and Ah(d)/Ah(d) mice that had been pretreated in vivo with benzo[a]pyrene. For all pyrolysis products tested, mutagenesis is 2-fold to more than 1000-fold greater with C57BL/6N and Ah(b)/Ah(d) than with DBA/2N or Ah(d)/Ah(d) liver fractions. A sucrose density gradient assay for detecting the Ah regulatory gene product, the receptor, was studied with C57BL/6N hepatic cytosol. At levels 100 times in excess of [1,6-(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin, nonlabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphthoflavone (inducers of cytochrome P(1)-450) are able to displace the radioligand from its hepatic cytosolic receptor; four pyrolysates from tryptophan, glutamic acid, and soybean globulin did not have this capacity. These data indicate that the pyrolysis products tested, although not effective as inducers of cytochrome P(1)-450, are most mutagenic when metabolized by P(1)-450. Potent P(1)-450 inducers-present in pyrolysates during the combustion process-might be present in quantities insufficient to initiate mutagenesis or carcinogenesis but might have a synergistic action, or act as "comutagens" or "cocarcinogens," with the N-containing heterocyclic pyrolysis products. A quantitative relationship between mutagenic and carcinogenic potency of these pyrolysis products remains, however, to be demonstrated.

Amino Acids↗
PubMed1978

delta9-Tetrahydrocannabinol and 17beta-estradiol bind to different macromolecules in estrogen target tissues.

delta9-Tetrahydrocannabinol (delta9-THC), added to the limit of its solubility, did not compete with tritiated 17beta-estradiol for binding to estrogen receptor sites in mouse mammary or uterine cytosols. On sucrose density gradients of low-ionic strength, mammary cytosol labeled with [3H]estradiol exhibited a binding peak near the "8S" region typical of estrogen receptor whereas in cytosol labeled with delta9-[3H]THC binding was limited to the nonspecific 4- to 5S region. Differences in sedimentation properties and reciprocal competition studies strongly refute previous claims that delta-9-THC binds to estrogen receptor and that by so doing it directly acts as an estrogen.

Animals↗
PubMed1978

Estrogen binding in mammary tissue of C3H mice with or without the mouse mammary tumor virus.

Substrains of C3H mice, with and without the milk-transmitted form of the mouse mammary tumor virus (MMTV)1, were compared to determine if the presence of MMTV altered estrogen action. Uterine weight response to oral diethylstilbestrol was the same for both substrains, (MMTV+ and MMTV-). The concentration of high-affinity estrogen binding sites ("receptors") in lactating mammary tissue did not differ significantly for MMTV+ versus MMTV- mice; the affinity of the estradiol-receptor interaction appeared higher, however, in cytosol from MMTV- mice. Sucrose density gradient analysis revealed no significant differences in sedimentation properties of cytosol receptor from the two substrains.

Animals↗
PubMed1976

In vivo and in vitro effects of dimethylbenz(a)anthracene on nuclear uptake of estrogen-receptor complex in rat uterus.

A single oral carcinogenic (20 mg) dose of 7,12-dimethylbenz(a)anthracene (DMBA) significantly reduced the uterine nuclear uptake of estrogen-receptor complex with immature Sprague-Dawley rats were injected with 0.05 microng of tritiated estradiol-17beta (E2) 24 hours after DMBA. Incubation experiments with whole uteri or with isolated uterine nuclei showed that DMBA in vitro does not act by reducing estrogen-receptor transformation or transport into nuclei. DMBA may act in vivo by stimulating hepatic degradation of injected estradiol-17beta so that less active form of the steroid reaches target tissues.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1975

Protection by 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) against mammary tumors and leukemia during prolonged feeding of 7,12-dimethylbenz(a)anthracene to female rats.

Female Sprague-Dawley rats, 36 days old, were pretreated for 2 weeks either with 100 ppm 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) or 250 ppm S-(1,2-dicarbeth-oxyethyl)OO-dimethyldithiophosphate (Malathion) in the diet. From day 50 they were given, via stomach tube, 21 consecutive daily doses of 0.714 mg 7,12-dimethylbenz[a]anthracene (DMBA). Pesticide diets and observation of the animals for mammary tumors continued until necropsy, 230 days after the start of DMBA administration. DDT-treated rats had a significantly lower mammary tumor incidence, prolonged tumor latency period, and fewer tumors per rat than did the control group. Animals given Malathion (an organophosphate pesticide) had a higher mammary tumor incidence, shortened latency period, more tumors per rat, and more actively growing tumors than did the control group (DMBA only). Leukemia incidence in rats surviving to necropsy (230 days after the start of DMBA administration) was 11/20 for control, 2/29 for DDT, and 8/12 for Malathion-treated rats. Leukemia was primarily myelogenous. DDT may inhibit DMBA-induced mammary tumors and leukemia by stimulating hepatic metabolism and excretion of DMBA so that less carcinogen is available to peripheral tissues. Malathion may potentiate DMBA induction of mammary tumors and leukemia by inhibiting the same enzyme systems induced by DDT.

9,10-Dimethyl-1,2-benzanthracene↗