Pia Larsson

Extrahepatic bioactivation of aflatoxin B1 in fetal, infant and adult rats

Whole-body autoradiography of 3H-labelled aflatoxin B1 (3H-AFB1) in female non-pregnant adult and infant Sprague-Dawley rats showed retention of tissue-bound radioactivity, in addition to the liver, in the mucosa and some glands in the nose, and in the mucosa of the nasopharynx, trachea, bronchioles, colon and caecum. The extrahepatic binding was most pronounced in the infant rats. In a rat pretreated with the glutathione (GSH)-depleting agent phorone, bound labelling was also seen in the superficial part of the mucosa of the glandular stomach. Autoradiography of 3H-AFB1 in pregnant rats showed a marked localization of bound AFB1-metabolites in the fetal nasal olfactory and tracheal mucosa. In vitro experiments demonstrated that the nasal olfactory mucosa had a much higher capacity than the liver to form AFB1-metabolites which bound to DNA and protein. The bioactivation was observed both pre- and post-natally and increased with age. Bioactivation was found also in the caecum, the colon and the lateral nasal gland (Stenos gland), but not in the small intestine, oesophagus or Harderian gland. Our results indicated that glutathione-S transferase activity catalysing the AFB1-8,9-epoxide GSH-conjugation was present in the nasal olfactory mucosa and liver at all pre- and post-natal ages examined. Several of the extrahepatic tissues able to bioactivate AFB1 have been reported to be targets for the carcinogenicity of the substance. Our results indicate that the extrahepatic carcinogenicity of AFB1 is correlated to a local bioactivation in the sensitive tissues.

Cell-specific activation of aflatoxin B1 correlates with presence of some cytochrome P450 enzymes in olfactory and respiratory tissues in horse

Horses may be exposed to aflatoxin B(1) (AFB(1)) via inhalation of mouldy dust, leading to high exposure of olfactory and respiratory tissues. In the present study the metabolic activation of AFB(1) was examined in olfactory and respiratory tissues in horse. The results showed covalent binding of AFB(1)-metabolites in sustentacular cells and cells of Bowmans glands in the olfactory mucosa, in some cells of the surface epithelium of nasal respiratory, tracheal, bronchial and bronchiolar mucosa and in some glands in these areas. Immunohistochemistry revealed that cells expressing proteins reacting with CYP 3A4- and CYP 2A6/2B6-antibodies had a similar distribution as those having capacity to activate AFB(1). Our data indicate that the cell-specific activation of AFB(1) correlates with presence of some CYP-enzymes in olfactory and respiratory tissues in horse.

Cellular activation and neuronal transport of intranasally instilled benzo(a)pyrene in the olfactory system of rats

Nasal tissues can be exposed to benzo(a)pyrene (BaP), e.g. present in diesel exhaust particles and some workplace atmospheres. In this study rats were given 3H-BaP intranasally. Autoradiography and beta-spectrometry were then used to trace cells in the nasal olfactory mucosa having capacity to activate the compound to tissue-bound metabolites. We also examined if deposition of 3H-BaP on the olfactory mucosa results in translocation of labelled material to the brain along olfactory neurons. The results showed that intranasal administration of 3H-BaP results in formation of tissue-bound metabolites in sustentacular cells and in the cells of Bowmans glands. Initially the bound material was localised to a higher extent to the sustentacular cells than to the cells of Bowmans glands, whereas at longer survival intervals the uptake in the cells of Bowmans glands dominated. In the latter the covalently bound material was accumulated to a higher extent in the nuclei than in the cytoplasms. We speculate that BaP may interact with the aryl hydrocarbon receptor (AhR) in these cells and that AhR may target activated BaP to the nucleus. Our results further indicated that application of 3H-BaP on the nasal mucosa results in transport of BaP and/or BaP-metabolites along the axons of the olfactory neurons to the olfactory bulb.

Autoradiographic studies of [3H]zearalenone in mice

Whole body autoradiograms of male, spayed and pregnant female mice after injection of [3H]zearalenone showed a rapid excretion of radioactivity into the bile as well as the urine. Specific localization was found in oestrogen target organs as the uterus, interstitial cells of the testicle and the follicles of the ovary. Unlike natural oestrogens zearalenone and/or its metabolites were not found in the adrenal cortex and bronchi of the lungs. Detection of radioactivity in an increased amount of fluid in the pleural and peritoneal cavities suggests an additional site of action of zearalenone. In the pregnant mice used, radioactivity was found only in the foetuses of late pregnancy mainly in the kidney, bile and connective tissue.

P-glycoprotein in intestines, liver, kidney and lymphocytes in horse.

P-glycoprotein (P-gp) is an important drug transporter, which is expressed in a variety of cells, such as the intestinal enterocytes, the hepatocytes, the renal tubular cells and the intestinal and peripheral blood lymphocytes. We have studied the localization and the gene and protein expression of P-gp in these cells in horse. In addition we have compared the protein sequence of P-gp in horse with the protein sequences of P-gp in several other species. Real time RT-PCR and Western blot showed gene and protein expression of horse P-gp in all parts of the intestines, but there was no strict correlation between these parameters. Immunohistochemistry showed localization of P-gp in the apical cell membranes of the enterocytes and, in addition, staining was observed in the intestinal intraepithelial and lamina propria lymphocytes. Peripheral blood lymphocytes also stained for P-gp, and gene and protein expression of P-gp were observed in these cells. There was a high gene and protein expression of P-gp in the liver, with P-gp-immunoreactivity in the bile canalicular membranes of the hepatocytes. Gene and protein expression of P-gp were found in the kidney with localization of the protein in different parts of the nephrons. Protein sequence alignment showed that horse P-gp has two amino acid insertions at the N-terminal region of the protein, which are not present in several other species examined. One of these is a 99 amino acid long sequence inserted at amino acid positions 23-121 from the N-terminal. The other is a six amino acid long sequence present at the amino acid positions 140-145 from the N-terminal. The results of the present study indicate that P-gp has an important function for oral bioavailability, distribution and excretion of substrate compounds in horse.

Expression and localization of BCRP, MRP1 and MRP2 in intestines, liver and kidney in horse

The gene and protein expression and the cellular localization of the ABC transport proteins breast cancer resistance protein (BCRP), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance-associated protein 2 (MRP2) have been examined in the intestines, liver and kidney in horse. High gene and protein expression of BCRP and MRP2 were found in the small intestines, with cellular localization in the apical membranes of the enterocytes. In the liver, MRP2 was present in the bile canalicular membranes of the hepatocytes, whereas BCRP was localized in the cytoplasm of hepatocytes in the peripheral parts of the liver lobuli. In the kidney both BCRP and MRP2 were predominantly present in the distal tubuli and in the loops of Henle. In most tissues, the gene and protein expression of MRP1 were much lower than for BCRP and MRP2. Immunostaining of MRP1 was detectable only in the intestines and with localization in the cytoplasm of enterocytes in the caecum and colon and in the cells of serous acini of Brunners glands in the duodenum and the upper jejunum. The latter cells were also stained for BCRP, but not for MRP2. Many drugs used in horse are substrates for one or more of the ABC transport proteins. These transporters may therefore have important functions for oral bioavailability, distribution and excretion of substrate compounds in horse.

Tracing tissues with 4-ipomeanol-metabolizing capacity in rats

Rats were given 3H-labelled 4-ipomeanol intravenously and whole-body autoradiography with freeze-dried sections, or with sections extracted with trichloroacetic acid, water and organic solvents, was performed to examine the disposition of unbound and bound radioactivity in various tissues. Microautoradiography with glutaraldehyde-fixed, resin-embedded material was used to investigate the cellular distribution of bound metabolites. Based on the data obtained from these experiments in vitro incubations with tissue-slices were carried out to examine the capacity by various tissues to form tissue-bound 3H from the 3H-labelled 4-ipomeanol and autoradiography of isolated organs after incubation with 3H-labelled 4-ipomeanol was performed to study the localization of radioactivity under in vitro conditions. The results showed a high formation of tissue-bound 3H in the lung in vitro and a localization of bound metabolites in several structures of the lung in vivo. In vitro formation of tissue-bound 3H was also found in the nasal olfactory and respiratory mucosa, the hard palate, the trachea, the liver and the kidney and this was also correlated with a localization of bound 3H in these tissues in vivo. Incubations of the lung, the nasal olfactory mucosa, the hard palate and the liver in CO- or N2-atmospheres or in the presence of the cytochrome P-450-inhibitor metyrapone showed decreased formation of tissue-bound 3H from the 3H-labelled 4-ipomeanol, indicating a role of cytochrome P-450 in the metabolism of 4-ipomeanol in the various tissues. The correlation between the in vitro capacity of various tissues to metabolize the 4-ipomeanol and the in vivo accumulation of tissue-bound metabolites in the same tissues indicate that a local bioactivation of the 4-ipomeanol takes place in these tissues in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential gene expression of CYP3A isoforms in equine liver and intestines.

Recently, seven CYP3A isoforms - CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 - have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin-isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K(m) for the LIPA substrate in the liver and the intestine, while the maximum velocity (V(max)) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.

Tissue uptake, distribution and elimination of 14C-PFOA in zebrafish (Danio rerio)

Perfluorooctanoic acid (PFOA) is a long-chain perfluorinated chemical that has been shown to be non-degradable and persistent in the environment. Laboratory studies on bioconcentration and compound-specific tissue distribution in fish can be valuable for prediction of the persistence and environmental effects of the chemicals. In the present study male and female zebrafish (Danio rerio) were continuously exposed to 10μg/L of radiolabeled perfluorooctanoic acid ((14)C-PFOA) for 40 days, after which the exposed fish were transferred to fresh clean water for another 80 days wash-out period. At defined periodic intervals during the uptake and wash-out, fish were sampled for liquid scintillation counting and whole body autoradiography to profile the bioconcentration and tissue distribution of PFOA. The steady-state concentration of (14)C-PFOA in the zebrafish was reached within 20-30 days of exposure. The concentration-time course of (14)C-PFOA displayed a bi-exponential decline during washout, with a terminal half-life of approximately 13-14 days. At steady-state the bioconcentration of (14)C-PFOA into whole-body fish was approximately 20-30 times greater than that of the exposure concentration, with no differences between females and males. The bioconcentration factors for liver and intestine were approximately 100-fold of the exposure medium, while in brain, ovary and gall bladder the accumulation factors were in the range 15-20. Whole-body autoradiograms confirmed the highest labeling of PFOA in bile and intestines, which implies enterohepatic circulation of PFOA. The (14)C-PFOA was also observed in maturing vitellogenic oocytes, suggesting chemical accumulation via yolk proteins into oocytes with plausible risk for adverse effects on early embryonic development and offspring health. The bioconcentration at several (14)C-PFOA exposure concentrations were also investigated (0.3-30μg/L). This showed that bioconcentration increased linearly with tank exposure in the present in vivo model under steady-state conditions. From this model tissue concentrations of PFOA can be predicted when the external exposure level is known. The present study has generated experimental data on PFOA kinetics in zebrafish that can be valuable for aquatic environmental risk assessment.

The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury.