Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon.

This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon, on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague Dawley rats. Rats were dosed with the conazoles at three dose levels by gavage for 14 days: myclobutanil (150, 75, and 10mgkg(-1) body weight day(-1); triadimefon (115, 50, and 10 mg kg(-1) body weight day-'), which included their maximum tolerated dose levels (MTD). Both myclobutanil and triadimefon significantly induced pentoxyresorufin O-depentylase activities at their MTD levels: myclobutanil, 8.1-fold at 150mgkg(-1) body weight day- ; and triadimefon, 18.5-fold at 115mgkg(-1) body weight day-'. Benzyloxyresorufin O-debenzylase activities were similarly increased: myclobutanil, 13.3-fold; triadimefon, 27.7-fold. Quantitative real-time reverse-transcription polymerase chain reaction assays were used to characterize the mRNA expression of specific CYP genes induced by these two conazoles. Myclobutanil and triadimefon treatment at their MTD levels significantly increased rat hepatic mRNA expression of CYP2B1 (14.3- and 54.6-fold), CYP3A23/3A1 (2.2- and 7.3-fold), and CYP3A2 (1.5- and 1.7-fold). Western immunoblots of rat hepatic microsomal proteins identified significantly increased levels of CYP isoforms after myclobutanil or triadimefon treatment at their MTD levels: CYP2BI/2 (4.8- and 5.3-fold), and CYP3A1 (2.2- and 2.9-fold). Triadimefon also increased CYP3A2 immunoreactive protein levels 1.8-fold. These results indicate that triadimefon and myclobutanil, like other triazole-containing conazoles, induced CYP2B and CYP3A families of cytochromes in rat liver.

Gene induction studies and toxicity of chemical mixtures.

As part of its mixtures program, the Agency for Toxic Substances and Disease Registry (ATSDR) supports in vitro and limited in vivo toxicity testing to further our understanding of the toxicity and health effects of chemical mixtures. There are increasing concerns that environmental chemicals adversely affect the health of humans and wildlife. These concerns have been augmented by the realization that exposure to chemicals often occurs to mixtures of these chemicals that may exhibit complex synergistic or antagonistic interactions. To address such concerns, we have conducted two studies with techniques that are being used increasingly in experimental toxicology. In the first study, six organochlorine pesticides (4,4 -DDT, 4,4 -DDD, 4,4 -DDE, aldrin, dieldrin, or endrin) were selected from the ATSDR Comprehensive Environmental Response, Compensation and Liability Act of 1980 (or Superfund) priority list and tested for their ability to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were dosed with and without selected environmental chemicals either individually or in defined combinations. Estradiol consistently elicited 10- to 23-fold dose-dependent inductions in this assay. By contrast, all six of the organochlorine pesticides showed no detectable dose-related response when tested either individually or in binary combinations. Thus, these chemicals as binary mixtures do not exhibit any additional estrogenicity at the levels tested in these assays. In the second study, arsenic [As(V)], cadmium [Cd(II)], chromium [Cr(III, VI)], and lead [Pb(II)] were tested in a commercially developed assay system, CAT-Tox (L), to identify metal-responsive promoters and to determine whether the pattern of gene expression changed with a mixture of these metals. This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal-transduction pathways. Singly, As(V), Cd(II), Cr(III, VI), and Pb(II) produced complex induction profiles in these assays. However, no evidence of synergistic activity was detected with a mixture of Cd(II), Cr(III), and Pb(II). These results have shown metal activation of gene expression through several previously unreported signal-transduction pathways and thus suggest new directions for future studies into their biochemical mechanisms of toxicity. In conclusion, the (italic)in vitro(/italic) methods used in these studies provide insights into complex interactions that occur in cellular systems and could be used to identify biomarkers of exposure to other environmental chemical mixtures.

Donor milk: what's in it and what's not.

Breastfeeding and human milk are widely recognized as optimal for human infants. However, if donor milk is used when mother's own milk is not available, some questions arise concerning the effects of storage, handling, and heat processing on the unique components of human milk. Holder pasteurization (62.5 degrees C for 30 minutes) of banked human milk is the method of choice to eliminate potential viral contaminants such as human immunodeficiency virus, human T-lymphoma virus, and cytomegalovirus, as well as tuberculosis and other bacterial contaminants, while maintaining the greatest possible complement of its unique bioactive factors. This article reviews some of the critical components of human milk and what is currently known about the effects of Holder pasteurization on their biological activity.

Effects of arsenic, cadmium, chromium, and lead on gene expression regulated by a battery of 13 different promoters in recombinant HepG2 cells.

Toxic metals occur naturally at low concentrations throughout the environment, but are found in higher concentrations at many of the hazardous waste sites on the EPA Superfund list. As part of the Agency for Toxic Substances and Disease Registry (ATSDR) mandate to evaluate the toxicity of metals and mixtures, we chose four of the high-priority metal pollutants from ATSDR's HAZDAT list, including arsenic, cadmium, chromium, and lead, to test in a commercially developed assay system, CAT-Tox(L) (Xenometrix). This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal transduction pathways. Our specific aims were to identify metal-responsive promoters and determine whether the pattern of gene expression changed with a mixture of metals. Humic acid was used in all assays as a carrier to help solubilize the metals and, in all cases, the cells were exposed to the humic acid-metal mixture for 48 h. Humic acid alone, at 50-100 microM, showed moderate activation of the XRE promoter, but little other notable activity. As(V), at doses of 50-250 microM, produced a complex profile of activity showing significant dose-dependent induction of the hMTIIA, GST Ya, HSP70, FOS, XRE, NFkappaBRE, GADD153, p53RE, and CRE promoters. Pb(II) showed dose-related induction of the GST Ya, XRE, hMTIIA, GRP78, and CYP IA1 promoters at doses in the range of 12-100 microM. Cd(II), at 1.25-15 microM, yielded significant dose-dependent induction of hMTIIA, XRE, CYP IA1, GST Ya, HSP70, NFkappaBRE, and FOS. Whereas Cr(III) yielded small, though significant inductions of the CRE, FOS, GADD153, and XRE promoters only at the highest dose (750 microM), Cr(VI) produced significant dose-related inductions of the p53RE, FOS, NFkappaBRE, XRE, GADD45, HSP70, and CRE promoters at much lower doses, in the range of 5-10 microM. Assays testing serial dilutions of a mixture comprising 7.5 microM Cd(II), 750 microM Cr(III), and 100 microM Pb(II) (the combination of metals most frequently found at National Priority List sites) showed significant dose-dependent induction of the hMTIIA promoter, but failed to show dose-related induction of any other promoter and showed no evidence of synergistic activation of gene expression by the metals in this mixture. Our results thus show metal activation of gene expression through several previously unreported signal transduction pathways, including As(V) induction of GST Ya, FOS, XRE, NFkBRE, GADD153, p53RE, and CRE; Pb(II) induction of GST Ya, XRE, Cyp IA1, and GADD153; Cd(II) induction of NFkBRE, Cyp IA1, XRE, and GST Ya; and Cr(VI) induction of p53RE, XRE, GADD45, HSP70, and CRE promoters, and thus suggest new insights into the biochemical mechanisms of toxicity and carcinogenicity of metals. It is also an important finding that no evidence of synergistic activity was detected with the mixture of Cd(II), Cr(III), and Pb(II) tested in these assays.

Six high-priority organochlorine pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells.

There have been increasing concerns that environmental chemicals may adversely affect the health of humans and wildlife by acting as endocrine modulators. These concerns have been augmented by the realization that human exposure occurs not just to single chemical agents, but typically to mixtures of chemicals that could exhibit estrogenic activity qualitatively and/or quantitatively different from that of individual components. To address these concerns, we have evaluated the ability of six organochlorine pesticides (4, 4'-DDT, 4,4'-DDD, 4,4'-DDE, aldrin, dieldrin, or endrin, all classified high priority by ATSDR) to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were exposed to these pesticides individually and in defined combinations. While estradiol consistently elicited 10- to 23-fold dose-dependent inductions in these assays, the six organochlorine pesticides showed no detectable dose-related response when tested individually. When tested in binary combinations, the pesticide mixtures showed no additional estrogenicity. Thus, the pesticides tested, singly or as mixtures, showed virtually no evidence of estrogenicity.

Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays.

Because of rampant concern that estrogenic chemicals in the environment may be adversely affecting the health of humans and wildlife, reliable methods for detecting and characterizing estrogenic chemicals are needed. It is important that general agreement be reached on which tests to use and that these tests then be applied to the testing of both man-made and naturally occurring chemicals. As a step toward developing a comprehensive approach to screening chemicals for estrogenic activity, three assays for detecting estrogenicity were conducted on 10 chemicals with known or suspected estrogenic activity. The assays were 1) competitive binding with the mouse uterine estrogen receptor, 2) transcriptional activation in HeLa cells transfected with plasmids containing an estrogen receptor and a response element, and 3) the uterotropic assay in mice. The chemicals studied were 17 beta-estradiol, diethylstilbestrol, tamoxifen, 4-hydroxytamoxifen, methoxychlor, the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), endosulfan, nonylphenol, o,p'-DDT, and kepone. These studies were conducted to assess the utility of this three-assay combination in the routine screening of chemicals, or combinations of chemicals, for estrogenic activity. Results were consistent among the three assays with respect to what is known about the estrogenic activities of the chemicals tested and their requirements for metabolic activation. By providing information on three levels of hormonal activity (receptor binding, transcriptional activation, and an in vivo effect in an estrogen-responsive tissue), an informative profile of estrogenic activity is obtained with a reasonable investment of resources.

Induced gene transcription: implications for biomarkers.

Numerous xenobiotics regulate cellular functions by altering transcription of target genes. Use of sensitive and specific biomarkers based on gene transcript levels may help clarify the shape of the dose-response curve in the low-dose region associated with human exposures to environmental concentrations of chemicals. We have quantified gene transcription induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in both animal models and humans with the use of Northern analysis and PCR-based methods. In addition, we describe a rapid and sensitive in vitro assay that we have used to screen chemicals and biological samples for their ability to alter gene transcription. Whereas some of the endpoints in our studies such as cytochrome P-450 1A1 are predictive indicators of exposure and dose, other gene responses such as growth factors are more complex and represent a critical event, progression, or adaptation to a pathological alteration. In conclusion, measurement of toxicant-induced gene transcription will contribute to the usefulness of biomarkers in addressing issues of human health and environmentally induced disease.

Modulation of steroid receptor-mediated gene expression by vitamin B6.

Gene transcription mediated by steroid hormones has become one of the most extensively characterized model systems for studying the regulation of gene expression in eukaryotic cells. However, specific details of gene regulation by steroid hormones are often complex and may be unique in specific cell types. Diverse regulatory mechanisms leading to either activation or repression of particular genes frequently involve interactions between steroid hormone receptors and other ubiquitous and/or cell-specific transcription factors that act on the complex promoter of the regulated gene. Interplay between steroid receptor-mediated and other signal transduction pathways may also be involved. In addition, recent novel results indicate that moderate variations in the intracellular concentration of pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, can have pronounced modulatory effects on steroid-induced gene expression. Specifically, elevation of intracellular PLP levels leads to decreased transcriptional responses to glucocorticoid, progesterone, androgen, or estrogen hormones. Conversely, cells in a vitamin B6-deficient state exhibit enhanced responsiveness to steroid hormones. One aspect of the mechanism by which these transcriptional modulatory effects of PLP occur has recently been shown to involve interruption of functional interactions between steroid hormone receptors and the nuclear transcription factor NF1. These findings--that the vitamin B6 nutritional status of cells modulates their capacity to respond to steroid hormones--impose an additional level of cell-specific control over steroid hormone regulation of gene expression and will serve as the focal point for this review.

Affinity of interactions between human glucocorticoid receptors and DNA: at physiologic ionic strength, stable binding occurs only with DNAs containing partially symmetric glucocorticoid response elements.

Sucrose density gradient shift assays were adapted to permit determination of the affinity of interaction between human glucocorticoid receptors (GR) and DNA under conditions of DNA excess. Saturation analyses were performed to ascertain dissociation constants for the interaction of activated human GR with each of five DNA fragments. Centrifugation of GR-DNA complexes on sucrose gradients under nearly isotonic salt conditions revealed similar affinities with dissociation constants in the range of 2-16 nM for GR interaction with DNA fragments containing glucocorticoid response elements (GREs) exhibiting partial dyad symmetry. By contrast, GR exhibited virtually no affinity for non-GRE-containing DNA or for DNA containing only GRE half-sites. Additionally, GR showed evidence of multiple-site interaction with a DNA fragment containing two partially symmetric GREs, but interacted at only one site of an MMTV LTR DNA fragment containing a single partially symmetric GRE along with a cluster of three half-GREs. Together these data indicate that under physiologically relevant conditions, glucocorticoid receptors have high selectivity and affinity only for DNA containing specific partially symmetric GREs and further suggest that this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid-responsive cells.

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

pBR322 contains glucocorticoid regulatory element DNA consensus sequences.

A computer search of the pBR322 DNA sequence identified five sites matching reported glucocorticoid regulatory element (GRE) DNA consensus sequences and three related sites. A pBR322 DNA fragment containing one GRE site was shown to bind immobilized HeLa S3 cell glucocorticoid receptor and to compete for receptor binding in a competitive binding assay. Conversely, a pBR322 DNA fragment devoid of GRE sites showed barely detectable interaction with glucocorticoid receptor in either of these assays. These results demonstrate the importance of GRE consensus sequences in glucocorticoid receptor interactions with DNA, and further identify a cause for high background binding observed when pBR322 DNA is used as a negative control in studies of glucocorticoid receptor-DNA interactions.

Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA.

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0 degrees C and in the nucleus and cytosol of cells incubated at 37 degrees C; binding of the MMTV DNA to highly purified receptor; and absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.

In vitro translation of major abundant class poly(A+)-mRNA from rat seminal vesicle.

Total poly (A)-containing messenger RNA [poly(A)-mRNAT] was isolated from seminal vesicles of adult male rats. By preparative 5%-20% linear sucrose gradient centrifugation in 1% SDS, a single peak was seen in the 11S region [poly(A)-mRNA11S]. Poly (A)-mRNAT and poly(A)-mRNA11S were translated in the wheat germ S-30 in vitro translation system. Laemmli-SDS-polyacrylamide slab gel electrophoresis of translation products of poly(A)-mRNAT revealed three major protein bands at approximately 18,500, 15,000, and 14,000 daltons. In vitro translation of poly(A)-mRNA11S across the 11S peak revealed partial purification of the mRNAs for the three predominant proteins. The lower molecular weight part of the 11S peak enriched for the mRNAs coding for the 15,000 and 14,000 dalton polypeptides, and the higher molecular weight side of the peak enriched for the 18,500 dalton mRNA. The highly abundant class of mRNA from rat seminal vesicle can be purified with one sucrose gradient centrifugation.

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA.

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images