Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing.

Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance sensing technique was used to monitor cell adhesion/spreading, barrier function and wound healing. Primary bronchial epithelium was compared with airway epithelial cell lines 16HBE14o-, BEAS-2B, NCI-H292 and A549. BEAS-2B, A549 and primary cells form a confluent monolayer more rapidly than do 16HBE14o- cells. In contrast, 16HBE14o- cells form stronger intercellular contacts, with a 10-fold higher resistance than BEAS-2B, A549 and NCI-H292 cells and a five-fold increase over primary cells. Accordingly, expression of the adhesion molecules zona occludens-1 and E-cadherin was highest in 16HBE14o- cells. These molecules were localised in intercellular junctions in both 16HBE14o- and primary cells. Finally, restoration of barrier function upon injury was impaired in BEAS-2B compared to 16HBE14o- cells. In conclusion, epithelial cell types display remarkable phenotypic differences and should, accordingly, be used to address specific research questions. 16HBE14o- cells appear most suitable for studies on barrier formation, whereas resemblance in attachment of primary and BEAS-2B and A549 cells makes the latter more important for translational research on cell-matrix contact.

Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD.

BACKGROUND: Goblet cell metaplasia, a common feature of chronic obstructive pulmonary disease (COPD), is associated with mucus hypersecretion which contributes to the morbidity and mortality among patients. Transcription factors SAM-pointed domain-containing Ets-like factor (SPDEF) and forkhead box protein A2 (FOXA2) regulate goblet cell differentiation. This study aimed to (1) investigate DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation and (2) compare this in airway epithelial cells from patients with COPD and controls during mucociliary differentiation. METHODS: To assess DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation, primary airway epithelial cells, isolated from trachea (non-COPD controls) and bronchial tissue (patients with COPD), were differentiated by culture at the air-liquid interface (ALI) in the presence of cytokine interleukin (IL)-13 to promote goblet cell differentiation. RESULTS: We found that SPDEF expression was induced during goblet cell differentiation, while FOXA2 expression was decreased. Importantly, CpG number 8 in the SPDEF promoter was hypermethylated upon differentiation, whereas DNA methylation of FOXA2 promoter was not changed. In the absence of IL-13, COPD-derived ALI-cultured cells displayed higher SPDEF expression than control-derived ALI cultures, whereas no difference was found for FOXA2 expression. This was accompanied with hypomethylation of CpG number 6 in the SPDEF promoter and also hypomethylation of CpG numbers 10 and 11 in the FOXA2 promoter. CONCLUSIONS: These findings suggest that aberrant DNA methylation of SPDEF and FOXA2 is one of the factors underlying mucus hypersecretion in COPD, opening new avenues for epigenetic-based inhibition of mucus hypersecretion.

First-pass metabolism of pentamethylmelamine in the rat liver.

The disposition of pentamethylmelamine (PMM) was studied in the male Wistar rat. PMM (5 mg/kg) was administered intraarterially, i.v. (5 and 10 mg/kg), via the portal vein, and into the duodenum to cannulated and unanesthetized rats (n greater than or equal to 4) via infusion. Parent compound and metabolites were quantified by gas chromatography. The areas under the plasma concentration-time curves of PMM after intraarterial and i.v. administration were equal and twice as large as the areas after portal vein and intraduodenal administration. This indicated insignificant lung metabolism for PMM; the low bioavailability of PMM when given via the portal vein or intraduodenally (in both cases, some 50% of an i.v. dose) was the result of presystemic metabolism in the liver. PMM was completely absorbed after intraduodenal administration, and no intestinal metabolism was observed. Linear kinetic behavior of i.v. PMM was observed in the 5- to 10-mg/kg dose range. The area under the plasma concentration-time curve of the first metabolite N2,N2,N4,N6-tetramethylmelamine was significantly greater when PMM was given via the portal vein or intraduodenally than when given intraarterially or i.v. This indicated either extrahepatic elimination/renal excretion of PMM or the existence of an additional metabolic pathway. However, experiments with adrenalectomized rats and rats with ligated blood flow to the kidneys did not alter the area for the first metabolite. These findings may be explained by the formation of unknown metabolites and/or reactive intermediates of PMM.

Low oral bioavailability of hexamethylmelamine in the rat due to simultaneous hepatic and intestinal metabolism.

The disposition of both hexamethylmelamine (HMM) after intraarterial, i.v., portal vein, and intraduodenal administration and of pentamethylmelamine following its i.v. administration was studied in male Wistar rats. HMM (5 and 10 mg/kg) and pentamethylmelamine (5 mg/kg) were infused via implanted cannulas into conscious animals (n greater than or equal to 4). Plasma levels of parent compound and of metabolites were determined by gas chromatography. The areas under the plasma concentration-time curves of HMM following its intraarterial and i.v. administration were not significantly different, indicating that HMM was not appreciably metabolized in the lung. Areas under plasma-concentration-time curves of HMM following portal vein and intraduodenal administration were 27 and 8% of the area under the plasma concentration-time curve after i.v. administration, respectively. Absorption of HMM was complete as judged from metabolite data. The reduced bioavailability of HMM intraduodenally was thus a consequence of presystemic elimination in the liver and the gut wall. Extraction ratios (or first-pass effects) of the liver and the gut wall were 73 and 71%, respectively. Linear kinetic behavior of HMM i.v. was observed in the 5- to 10-mg/kg dose range. Extensive gut wall metabolism may have important implications for the antitumor activity mechanism of HMM.

Cellular and subcellular studies of the biotransformation of hexamethylmelamine in rat isolated hepatocytes and intestinal epithelial cells.

The antitumor agent hexamethylmelamine is subject to oxidative metabolic conversion in rat isolated liver and small intestinal cells (conversion 40 times higher in hepatocytes). This N-demethylation is mediated by cytochrome P-450 in the microsomal fractions, and in mitochondrial preparations it has been found to occur via N- methylolpentamethylmelamine . Somehow, pentamethylmelamine, hydroxymethylpentamethylmelamine , or an intermediary metabolite becomes trapped in the intact cell, but the nature of the adduct formed is still unresolved. Pretreatment of rats with 3-methylcholanthrene p.o. caused a 5-fold increase in hexamethylmelamine turnover. Phorone administered in vivo prior to cell preparation (liver and gut) caused an increase in pentamethylmelamine production. The latter results together with results of adding glutathione to cell incubations demonstrate that glutathione contributes to the regulation of cytochrome P-450-mediated N-demethylation of hexamethylmelamine.

Kinetic properties of the rat intestinal microsomal 1-naphthol:UDP-glucuronosyl transferase. Inhibition by UDP and UDP-N-acetylglucosamine.

The kinetic properties of the rat intestinal microsomal 1-naphthol:UDPglucuronosyltransferase (EC 2.4.1.17) were investigated in fully activated microsomes prepared from isolated mucosal cells. The enzyme appeared to follow an ordered sequential bireactant mechanism in which 1-naphthol and UDP-glucuronic acid (UDPGlcUA) are the first and second binding substrates and UDP and 1-naphthol glucuronide the first and second products, respectively. Bisubstrate kinetic analysis yielded the following kinetic constants: Vmax = 102 +/- 6 nmol/min per mg microsomal protein, Km (UDPGlcUA) = 1.26 +/- 0.10 mM, Km (1-naphthol) = 96 +/- 10 microM and Ki (1-naphthol) = 25 +/- 7 microM. The rapid equilibrium random or ordered bireactant mechanisms, as well as the iso-Theorell-Chance mechanism, could be excluded by endproduct inhibition studies with UDP.UDP-N-acetylglucosamine (UDPGlcNAc), usually found to be an activator of UDP glucuronosyltransferase in liver microsomes, acted as a full competitive inhibitor towards UDPGlcUA in rat intestinal microsomes. With regard to 1-naphthol UDPGlcNAc exhibited a dual effect: both inhibition and activation was observed. The effect of activation by MgCl2 and Triton X-100 on the kinetic constants and the inhibition patterns of UDP and UDPGlcNAc were investigated. The results obtained suggest that latency in rat intestinal microsomes may be due to endproduct inhibition by UDP. This endproduct inhibition could be abolished by in vitro treatment with MgCl2 and Triton X-100.

Ca2+-free perfusion of isolated rat heart: early irreversible changes and discrepancy between functional impairments and release of cellular constituents.

The effects of repeated (10 X), brief Ca2+-free perfusions (20 to 120 s) on myocardial contractility, coronary flow and release of cellular constituents of isolated rat heart were investigated. Ten successive Ca2+-deprivations of 20 or 40 s had no irreversible influences on cardiac performance. Ca2+-deprivations, however, of 60 s or longer, resulted in a substantial depression of contractile activity during recovery, which effect was parallelled by the development of myocardial contracture. Both effects appeared to be additive in character (and thus irreversible) and were dependent upon the duration and number of Ca2+-deprivations. The effect on coronary flow was biphasic: after an initial, rapid increase coronary flow steadily declined and was almost normal at the end of the experiments. The release of cellular constituents into the coronary effluent only occurred during reperfusion with Ca2+ after Ca2+-free periods of 60 s or longer. This loss of cellular material from the heart was observed mainly after the first Ca2+-deprivation and was only small or even negligible during the subsequent Ca2+-deprivations. The overall release of cellular material was clearly dependent on the duration of the Ca2+-free period, but was badly related to the overall loss in contractile activity. It was concluded that a relatively brief Ca2+-free perfusion period may induce a small but irreversible damage to the heart. This damage becomes visible, and more deleterious, when the brief Ca2+-washout is repeated several times. In addition, it was concluded that Ca2+-free-induced functional impairments and the release of cellular constituents of the heart are badly correlated and may be caused by two separate, but probably interrelated, mechanisms.

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation.

A comparison is made between microsomal NADPH-dependent H2O2 production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H2O2 formation was observed during NADPH-dependent disposition (10 min) of 100 microM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 microM) and its mono-N-demethylated metabolite tofenacine (100 microM) decreased the H2O2 formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H2O2 production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.

The regulation of oxidative drug metabolism by growth hormone in the dwarf goat: differences from and similarities to the mechanisms in rats.

The effects of bovine GH (BST), administered in different dose patterns, on in-vivo oxidative drug metabolism, were studied in female dwarf goats. Animals received recombinantly derived methionyl BST at a dose of 500 micrograms/kg body weight per 24 h for 6 days. It was administered to one group of goats as one s.c. injection per day, another group received a similar 24-h dose divided into three s.c. injections given at 8-h intervals, and the third group received 50 micrograms BST/kg body weight every 2.5 h by a pulsative i.v. infusion. Oxidative metabolic capacity was assessed by determining plasma sulphadimidine (SDD) elimination and urinary metabolite excretion. SDD shows a marked sex-dependent plasma elimination in dwarf goats, with male goats having a lower plasma clearance than female goats. When BST was given by daily injection, no clear effects on SDD plasma clearance or urinary metabolite excretion were observed. However, when the total dose was divided into three injections given at 8-h intervals, the plasma SDD elimination rate decreased. This was associated with a decrease in urinary excretion of the two main hydroxy SDD metabolites. When BST was given by discontinuous i.v. infusion, simulating the male endogenous plasma GH pattern, a marked decrease in SDD plasma clearance was observed. In addition, the excretion of the two urinary hydroxy metabolites was considerably reduced. These results suggest that GH can affect drug oxidation in dwarf goats via mechanisms similar to those suggested for rats. However, in the dwarf goat, the sex differences in drug metabolism are opposite to those in rats.

Glucuronidation in the rat intestinal wall. Comparison of isolated mucosal cells, latent microsomes and activated microsomes.

Glucuronidation and sulphation of 1-naphthol and 7-hydroxycoumarin was studied in isolated rat intestinal epithelial cells and in microsomes prepared from these cells. In the isolated cells formation of 1-naphthol sulphate could not be detected. Sulphate conjugates of 7-hydroxycoumarin constitute a minor portion of total conjugates formed. Maximum glucuronidation rates for 1-naphthol and 7-hydroxycoumarin do not differ significantly from each other (approximately 12.5 nmoles/min X g intestine). The intestinal microsomal UDP-glucuronosyltransferase, prepared from isolated cells, could be activated in vitro by Triton X-100 and MgCl2. Activation increased both Kappm and Vmax for 1-naphthol; Kappm for UDP-glucuronic acid was decreased by activation with MgCl2 but increased again by further addition of Triton X-100. In fully activated microsomes Kappm for 1 naphthol was 69.7 +/- 13.9 microM and Vmax was 70.0 +/- 3.9 nmoles/min X mg microsomal protein; Kappm for UDP-glucuronic acid was 0.67 +/- 0.06 mM. The glucuronidation rate (expressed as nmoles/min X g intestine) in microsomes is substantially higher than in isolated cells. It appears that glucuronidation in intact cells is limited by factors other than the extracellular substrate concn. Both cellular uptake of the substrate and availability of UDP-glucuronic acid can play a significant role. It is concluded that isolated mucosal cells are more suitable for predicting intestinal first-pass metabolism of phenolic xenobiotics than intestinal microsomes, because cellular substrate uptake and cosubstrate availability appear to be important determinants of the maximum glucuronidation rate.

Prevention of oxidant-induced cell death in Caco-2 colon carcinoma cells after inhibition of poly(ADP-ribose) polymerase and Ca2+ chelation: involvement of a common mechanism.

The human colon carcinoma cell line Caco-2 was exposed to the oxidative stress-inducing agents menadione (MEN), 2,3-dimethoxy-1,4-naphthoquinone, and hydrogen peroxide. All three agents caused DNA damage which was assessed by alkaline unwinding. Further, all three agents induced intensive NAD+ depletion, followed by a decrease in intracellular ATP and viability. Inhibition of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) by 3-aminobenzamide prevented the depletion of NAD+. These cells had a higher viability and ATP content. The most pronounced effect was observed with 25 microM of MEN, while at higher levels a partial preservation of NAD+ was observed with no effect on ATP or viability. The chelation of intracellular calcium by bis-(o-aminophenoxy)-ethane-N,N,N1,N1-tetraacidic acid/tetraacetoxymethyl) ester also prevented the dramatic loss of NAD+, demonstrating that Ca2+ is an activating factor in PARP-mediated cell killing.

Quinone toxicity in DT-diaphorase-efficient and -deficient colon carcinoma cell lines.

The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.

Kinetics of in vitro O-deethylation of phenacetin and 7-ethoxycoumarin by rat intestinal mucosal cells and microsomes. The effect of induction with 3-methylcholanthrene and inhibition with alpha-naphthoflavone.

A novel, sensitive (0.5 ng) assay for acetaminophen, using HPLC with selective electro-chemical detection, enabled us to study rat small intestinal O-deethylation of phenacetin and compare it with corresponding 7-ethoxycoumarin-O-deethylation. Two in vitro systems, i.e. isolated intestinal mucosal cells and microsomal fractions thereof, were used to study kinetics for the O-deethylation of both substrates. Kapp m- and Vmax-values are similar for 7-ethoxycoumarin- and phenacetin-O-deethylation. Apparent Km-values varied between 50 and 70 microM in control rats and decreased after 3-methylcholanthrene pretreatment to 20-45 microM. Vmax-values were increased by 3-methylcholanthrene pretreatment. O-Deethylation was inhibited equally in cells and microsomes by alpha-naphthoflavone, but is inhibited more markedly in intestinal preparations after pretreatment with 3-methylcholanthrene. It is suggested that 7-ethoxycoumarin and phenacetin are O-deethylated by different forms of cytochrome P-450 with almost identical Kapp m and that these enzymes have a different distribution along the villus.

Distribution of glucuronidation capacity (1-naphthol and morphine) along the rat intestine.

The distribution of glucuronidation capacity along the rat intestine was investigated using mucosal cells, isolated from the small intestine, the caecum, and the colon plus rectum. The glucuronidation capacity for 1-naphthol decreases from 787 +/- 75 (duodenum) to 128 +/- 13 (colon plus rectum) pmoles/min X mg cell protein. The ratio between 1-naphthol and morphine glucuronidation was constant throughout the intestine (7.15 +/- 0.37). The distribution of maximal activity of UDP-glucuronosyltransferase in intestinal cell homogenates follows the same pattern. The maximal activity of UDPglucose dehydrogenase in homogenates corresponds closely to the glucuronidation rate in mucosal cells. The activity of beta-glucuronidase in intestinal cell homogenates is constant along the duodenum and jejunum but increases throughout the terminal ileum, caecum, colon and rectum. Subcellular fractionation studies using marker enzymes indicate that UDPglucose dehydrogenase and beta-glucuronidase are cytosolic enzymes in intestinal mucosal cells. Although UDP-glucuronosyltransferase activity is found in both the mitochondrial and the microsomal fractions, no indications for a mitochondrial localization of this enzyme can be found. Activity in the mitochondrial fraction appears to be due to endoplasmic reticulum, associated with the mitochondrial fraction.

Cytochrome P450 induction and metabolism of alkoxyresorufins, ethylmorphine and testosterone in cultured hepatocytes from goats, sheep and cattle.

Very little is known of cytochrome P450 (P450) patterns and enzyme characteristics in food-producing animal species. Oxidative metabolism of alkoxyresorufins, ethylmorphine (EtM) and testosterone (TST) was used to monitor the effects of the P450 inducers phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX) and triacetyloleandomycin (TAO) in primary cultured hepatocytes from goats, sheep and cattle. BNF effectively and specifically induced ethoxyresorufin deethylase (> 20-fold), indicating the presence of an inducible P450 1A form, and down-regulated EtM demethylation and most selected TST hydroxylations. In non-induced hepatocyte cultures, TST was metabolized to 6 beta-, 2 beta-, 12 beta-, and 11 alpha-hydroxy-TST (OHT). PB and, to a lesser extent, DEX non-specifically induced all OHT formations, and EtM demethylation. TAO almost completely inhibited OHT formation and EtM demethylation. These results indicate the involvement of principally one P450 form, or a restricted number of related P450 forms, presumably belonging to the P450 3A subfamily. In western blot analysis, cross reactivity was found with rat anti-P450 3A1 and anti-sheep P450 3A. A more specific PB effect was observed for 16 alpha-OHT, which may be formed though a ruminant P450 2B form. None of the inducers influenced pentoxyresorufin depentylase (PROD) or EtM O-deethylation. Metabolite patterns and inducibility of selected activities in ruminant hepatocytes are in accordance with previous findings in goats in vivo. Cytochrome P450 characteristics in ruminants appear to differ from those in rats whereas similarities to the situation in humans appear to exist.

Biotransformation of scoparone used to monitor changes in cytochrome P450 activities in primary hepatocyte cultures derived from rats, hamsters and monkeys.

The coumarin derivative scoparone is regioselectively demethylated yielding isoscopoletin and scopoletin. The ratio of the formation rates of these two metabolites (isoscopoletin/scopoletin; I/S ratio) is reported to mirror the contribution of several cytochrome P450 (P450) isoenzymes to the biotransformation of scoparine. The metabolism of scoparine has been studied in primary liver cell cultures of rats, hamsters, cynomolgus monkeys and in human liver cells. Rat hepatocytes appeared to metabolize scoparone 7 to 10 times slower than those of hamsters and monkeys. In hepatocyte monolayers of all three species the loss of P450 was paralleled by a decrease in total scoparone metabolism. In hamsters but not in rats, a decrease of the I/S ratio was found during primary culture of liver cells. A similar shift in the metabolic pattern of scoparine observed with the monkey hepatocytes was statistically not significant. Most likely, in hamster and possibly in monkey hepatocyte cultures the different P450s involved in scoparone metabolism decrease at unequal rates. In rat liver cells, however, the pattern of these P450 isoenzymes remains more or less unaltered. In contrast to liver cells from the other species, human hepatocytes did not secrete scopoletin in detectable amounts. Scoparone demethylation in humans may be qualitatively different from that in other mammals.

Microsomal lauric acid hydroxylase activities after treatment of rats with three classical cytochrome P450 inducers and peroxisome proliferating compounds.

In order to investigate a proposed relationship between induction of hepatic microsomal lauric acid hydroxylase activity and peroxisome proliferation in the liver, male Wistar rats were treated with peroxisome proliferating compounds, and the lauric acid hydroxylase activity, the immunochemical detectable levels of cytochrome P450 4A1 and the activities of peroxisomal enzymes were determined. In addition, the levels of cytochrome P450 4A1 and lauric acid hydroxylase activities were studied after treatment of rats with three cytochrome P450 inducers. After treatment with aroclor-1254, phenobarbital or 3-methylcholanthrene total cytochrome P450 was 1.7-2.7 times induced. However, no induction of lauric acid omega-hydroxylase activities or P450 4A1 levels were found. After treatment of rats with di(2-ethylhexyl)phthalate (DEHP) a dose-dependent induction of lauric acid omega-hydroxylase activities, levels of cytochrome P450 4A1 and peroxisomal fatty acid beta-oxidation was found. Even at a dose-level of 100 mg DEPH/kg body weight per day a significant induction of these activities was observed. The main metabolites of DEHP, mono(2-ethylhexyl)phthalate and 2-ethyl-1-hexanol, also caused an induction of levels of P450 4A1, lauric acid omega-hydroxylase activities and the activity of peroxisomal palmitoyl-CoA oxidase. 2-Ethyl-1-hexanoic acid did not influence lauric acid omega-hydroxylase activities, but did induce levels of P450 4A1 and palmitoyl-CoA oxidase activities. Three other compounds (perfluoro-octanoic acid, valproate and nafenopin) induced both lauric acid omega-hydroxylase activity and peroxisomal palmitoyl-CoA oxidase activity. The plasticizer, di(2-ethylhexyl)adipate, did not induce levels of P450 4A1, lauric acid omega-hydroxylase activities or palmitoyl-CoA oxidase activities. With the compounds tested a close association between the induction of lauric acid omega-hydroxylase activities and peroxisomal palmitoyl-CoA oxidase activity was found. These data support the theory that peroxisome proliferating compounds do induce lauric acid omega-hydroxylase activities and that there might be a mechanistic inter-relationship between peroxisome proliferation and induction of lauric acid omega-hydroxylase activities.

Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey. Relationship between interspecies differences in biotransformation and peroxisome proliferating potencies.

Primary hepatocyte cultures derived from rat, rabbit, guinea pig and monkey have been treated in vitro with metabolites of di(2-ethylhexyl)phthalate, i.e. mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (metabolite V) and mono(2-ethyl-5-oxohexyl)phthalate (metabolite VI). In rat hepatocyte cultures MEHP and metabolite VI were equally potent in inducing peroxisome proliferation, while metabolite V was much less potent. In rat hepatocytes a 50% increase in both peroxisomal palmitoyl-CoA oxidase activity and microsomal lauric acid omega-hydroxylation activity was found after treatment with 5-15 microM MEHP. In guinea pig, rabbit and monkey hepatocyte cultures, a 50% increase in peroxisomal palmitoyl-CoA oxidase activity was found after treatment with 408-485 microM MEHP. No induction of lauric acid omega-hydroxylation activity was found. These results indicate that peroxisome proliferation can be induced by MEHP in rabbit, guinea pig and monkey hepatocytes, but that these species are at least 30-fold less sensitive to peroxisome proliferation induction than rats. The proposed mechanistic inter-relationship between induction of lauric acid omega-hydroxylation activity and peroxisome proliferation is found in rat hepatocytes, but not in hepatocytes of the other three species. Treatment of guinea pig hepatocyte cultures with MEHP resulted in an increase in triglyceride concentrations in the hepatocytes. In rat and rabbit hepatocyte cultures, triglyceride concentrations were much less altered by MEHP. In monkey hepatocytes a decrease in hepatic triglyceride concentration was found after treatment with MEHP. These effects are in agreement with in vivo effects observed before. After treatment of primary hepatocyte cultures with MEHP, high concentrations of omega- and (omega-1)-hydroxylated metabolites of MEHP were found in media from rat, rabbit and guinea pig cultures. The formation of these metabolites did not decline in time. During treatment the metabolite profile in media from rat hepatocyte cultures moved towards omega-hydroxy metabolites of MEHP. In media from monkey hepatocyte cultures the lowest concentrations of hydroxylated metabolites were determined. No major species differences were found in the potency to form oxidized MEHP metabolites, and thus no unique metabolite differences were found, which could explain the species differences in sensitivity for peroxisome proliferation.

Hepatic cytochrome P450 induction in goats. Effects of model inducers on the metabolism of alkoxyresorufins, testosterone and ethylmorphine, and on apoprotein and mRNA levels.

Male and female African dwarf goats were treated orally with phenobarbital (PB) or triacetyloleandomycin (TAO), or subcutaneously with beta-naphthoflavone (BNF). Hepatic microsomal cytochrome P450 content was increased by PB and TAO, but not by BNF. PB effects on P450 activities were non-selective: ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD), hydroxylation of testosterone (TST) and demethylation of ethylmorphine (ETM) were all induced by a factor of 2-3. A similar non-selective induction was observed with TAO, except for EROD and PROD (no effects). After PB and TAO treatment, increased levels of a protein cross-reactive with anti-sheep P450 3A and 2B were found. Thus, in dwarf goats, both PB and TAO appeared to be P450 3A inducers. Selective PB effects related to a P450 2B form on PROD are lacking but 16 alpha-hydroxylation of TST was induced markedly. At the mRNA level, PB induced an mRNA that showed good sequence homology with a human P450 3A4 cDNA probe, rather than with a rat 3A1 probe. BNF selectively induced EROD, whereas TST hydroxylation and ETM dealkylation were inhibited. With BNF-treated animals, increased concentrations of a protein cross-reactive with anti-rat P450 1A1/1A2 and of an mRNA that showed homology with a human 1A1 cDNA probe, but not with a mouse 1A1/1A2 probe, were observed.

Comparison of cytochrome P450 isoenzyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities.

The metabolic profile of seven subfamilies of cytochrome P450 (P450IA, IIA, IIB, IIC, IIE, IIIA, IVA) was studied in rat liver (in vivo) and in primary hepatocyte cultures (in vitro) after treatment with various inducers. The dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD), aniline 4-hydroxylation and the regio- and stereoselective hydroxylation of testosterone were measured to characterize the isoenzyme pattern in intact hepatocytes and in liver microsomes. Occurrence of isoenzyme apoproteins was determined using Western blotting. Primary cultures of rat hepatocytes retain the capacity to respond to inducers of isoenzymes belonging to six different subfamilies (P450IA, IIA, IIB, IIC, IIIA and IVA). Treatment of cells with beta-naphthoflavone revealed a P450-activity profile similar to in vivo, namely a highly induced EROD (P450IA1), a small enhancement of testosterone 7 alpha-hydroxylation (P450IIA) and a marked reduction in 2 alpha- and 16 alpha-hydroxylation (P450IIC11). Exposure of cultured cells to phenobarbital resulted in a higher testosterone 16 beta-hydroxylation (reflecting P450IIB), though to a lesser extent than in vivo. The induction of P450IIIA due to both phenobarbital and dexamethasone, as mirrored by 6 beta- and 15 beta-hydroxylation of testosterone, was the same in cultured hepatocytes and in vivo. Treatment of cells with clofibric acid resulted in an induction profile similar to the one observed in liver microsomes from clofibrate-treated rats: the apoprotein P450IVA as well as the apoprotein P450IIB1/2 and its associated activities (PROD and testosterone 16 beta-hydroxylation) were induced. Isoniazid, a known in vivo inducer of P450IIE1 and aniline 4-hydroxylation, did not change any of the determined P450-dependent activities in vitro.