Mary P. Scott

Studies on the induction of rat hepatic CYP1A, CYP2B, CYP3A and CYP4A subfamily form mRNAs in vivo and in vitro using precision-cut rat liver slices

1. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan®) was used to examine the induction of some selected rat hepatic cyto-chrome P450 (CYP) forms in vivo and in vitro using cultured precision-cut liver slices. 2. TaqMan primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1, CYP2B1/2, CYP3A1, CYP3A2 and CYP4A1 mRNAs. 3. To characterize the responsiveness of the rat CYP mRNA TaqMan primers and probe sets, rats were treated in vivo with a single intraperitoneal dose of 500 mg kg − 1 Aroclor 1254 (ARO) and with four daily oral doses of either 50 mg kg − 1 day − 1 dexamethasone (DEX) or 75 mg kg − 1 day − 1 methylclofenapate (MCP). Treatment with ARO produced 22 600-, 5480-, 648-, 52-, 47- and 9-fold increases in levels of CYP1A1, CYP2B1, CYP2B1/2, CYP1A2, CYP3A1 and CYP3A2 mRNA, respectively. DEX treatment produced 97-, 24-, 8- and 4-fold increases, respectively, in CYP3A1, CYP2B1, CYP2B1/2 and CYP3A2 mRNA levels, and MCP produced 339-, 126- and 25-fold increases, respectively, in CYP4A1, CYP2B1 and CYP2B1/2 mRNA levels. All three CYP inducers also increased microsomal CYP content and produced corresponding increases in CYP1A, CYP2B, CYP3A and CYP4A form marker enzyme activities. 4. Rat liver slices were cultured for 6 and 24 h in medium containing 0.1 µ M insulin and 0.1 µ M DEX, and also for 24 h in medium containing only 0.1 µ M insulin (DEX-free medium). Liver slices were cultured in control medium or in medium containing either 10 µ M β -naphthoflavone (BNF), 10 µ g ml − 1 ARO, 500 µ M sodium phenobarbitone (NaPB), 20 µ M pregnenolone-16 α -carbonitrile (PCN), 50 µ M Wy-14,643 (WY) or 50 µ M MCP. 5. With the exception of the effect of BNF on CYP1A1 mRNA levels, the induction of all the CYP mRNAs studied was greater after 24- than after 6-h treatment. Generally, the magnitude of induction of CYP mRNA levels was greater after 24 h in liver slices cultured in DEX-free than in DEX-supplemented medium. 6. Treatment of liver slices with BNF and ARO for 24 h in DEX-free medium produced 21- and 35-fold increases, respectively, and 38- and 37-fold increases, respectively, in CYP1A1 and CYP1A2 mRNA levels. NaPB, PCN, WY and MCP did not increase either CYP1A1 or CYP1A2 mRNA levels. 7. After 24 h, levels of CYP2B1/2 mRNA were increased 18-, 20-, 9-, 16- and 13-fold by treatment with ARO, NaPB, PCN, WY and MCP, respectively. PCN also produced 56- and 4-fold increases, respectively, in CYP3A1 and CYP3A2 mRNA levels. 8. Treatment with WY and MCP for 24 h produced 437- and 186-fold increases, respectively, in levels of CYP4A1 mRNA. None of the other CYP inducers studied had any effect on CYP4A1 mRNA levels. 9. The results demonstrate the utility of cultured precision-cut liver slices as an in vitro model system to evaluate the effects of xenobiotics on rat CYP1A, CYP2B, CYP3A and CYP4A form mRNA levels.

Association of c‐fos mRNA expression and excitotoxicity in primary cultures of mouse neocortical and cerebellar neurons

The effect of excitatory amino acids (EAAs) on c‐fos mRNA expression was studied in primary cultures of mouse cerebellar granule cells and in neocortical neurons after 2 and 7 days in vitro (div). In cultured granule cells at 2 and 7 div, and in cortical neurons at 2 div, exposure to low levels (≤10 μM) of a variety of EAAs (viz. glutamate [Glu], S‐sulpho‐L‐cysteine [SC], N‐methyl‐D‐aspartate [NMDA], α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole [AMPA], and kainate [KA]) resulted in a transient increase in the level of c‐fos mRNA which peaked at 30 min but returned to a basal level by 120 min. However, exposure of granule cells (7 div) to high levels (250 μM) of Glu, NMDA, KA, SC and of cortical neurons (7 div) to high levels (250 μM) of Glu, NMDA, KA, SC, or AMPA and to low levels (≤10 μM) of Glu and AMPA resulted in a delay in c‐fos mRNA induction but a subsequent, progressive increase that was sustained for at least 240 min. Furthermore, this effect was accompanied by a dose‐related increase in the release of the cytosolic enzyme, lactate dehydrogenase, used as an indicator of excitotoxicity. A ratio (Q240/30) for the steady‐state levels of c‐fos mRNA after 30 min and 240 min of exposure to EAAs was determined which showed that Q240/30 >2 correlated reproducibly with excitotoxic cell death, whereas a ratio of ≤1 correlated with a nonexcitotoxic event. In both cell types at 7 div, coadministration of the selective NMDA receptor antagonist, DL(±)‐2‐amino‐5‐phosphonopentanoic acid (APV) with cytotoxic levels of Glu 1) protected against EAA‐induced neurotoxicity and 2) exhibited a transient c‐fos mRNA expression (Q240/30 values ≈1). In contrast, the AMPA/KA receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), provided no protection against excitotoxicity and had no significant effect on the Glu‐induced delay in c‐fos mRNA expression. These results suggest that the Q240/30 c‐fos mRNA ratio may 1) be used as a predictive index for excitotoxic neuronal death, 2) provide information on the identity of the receptor subtype mediating excitotoxicity in different brain cell types, and 3) aid in establishing the role of excitotoxicity during the development of neurons in vitro. J. Neurosci. Res. 48:533–542, 1997.

Excitatory amino acid-induced cytotoxicity in primary cultures of mouse cerebellar granule cells correlates with elevated, sustained c-fos protoncogene expression

Abstract An elevated, sustained expression of c- fos mRNA was found in primary cultures of mouse cerebellar granule cells following exposure to toxic concentrations of the excitatory amino acids, l -glutamate, l -homocysteate, S -sulpho- l -cysteine and N -methyl- d -aspartate (NMDA), using leakage of lactate dehydrogenase (LDH) as an indicator of cytotoxicity. In contrast, when used at non-toxic concentrations these compounds induced a rapid and transient increase in c- fos mRNA levels. Both LDH release and elevated, sustained c- fos mRNA induction were blocked (in the case of l -homocysteate) or reduced (in the case of l -glutamate and S -sulpho- l -cysteine) by the selective NMDA receptor antagonist ( dl (±)-2-amino- 5-phosphonopentanoic acid) whereas 6-cyano-7-nitroquinoxaline-2,3-dione (a selective antagonist at non-NMDA ionotropic receptors) had no effect. These data suggest a role for altered c- fos mRNA expression in excitotoxic mechanisms.

Calcium influx via L‐type voltage‐gated channels mediates the delayed, elevated increases in steady‐state c‐fos mRNA levels in cerebellar granule cells exposed to excitotoxic levels of glutamate

The altered kinetics of steady‐state c‐fos mRNA production in cultured cerebellar granule cells under excitotoxic conditions was investigated in neurons subjected to depolarising stimuli, namely, high KCl and L‐glutamate (Glu), in which Ca2+ influx occurs by differing routes. Increases in intracellular‐free calcium levels ([Ca2+]i) stimulated by nontoxic or toxic levels of Glu were blocked by selective N‐methyl‐D‐aspartate (NMDA) receptor antagonism; were blocked only partially by the L‐type channel blocker, nifedipine; and were unaffected by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)/kainate receptor antagonists. Glu‐induced cell death was prevented only by NMDA receptor blockade. Exposure of cells to nontoxic levels of Glu resulted in a transient increase in c‐fos mRNA levels, whereas an excitotoxic dose produced a delay in the appearance of c‐fos mRNA but a subsequent, progressive, and sustained (>4 hr) increase. An excitotoxic dose of Glu in combination with either nifedipine or selective NMDA receptor antagonists resulted in the normal, transient increase of c‐fos mRNA levels. Chronic exposure to 55 mM KCl caused no cytotoxicity, although it resulted in a delayed, elevated increase in c‐fos mRNA levels that was unaffected by NMDA receptor blockade but reverted to the normal, transient profile of c‐fos mRNA formation when it was coadministered with nifedipine. The KCl‐induced increase in [Ca2+]i levels was inhibited dramatically by nifedipine but was unaffected by any of the ionotropic Glu receptor antagonists. The results support the notion that the appearance of a delayed but elevated increase in steady‐state c‐fos mRNA levels following exposure to excitotoxic doses of Glu is mediated specifically by calcium influx via L‐type voltage‐gated channels. J. Neurosci. Res. 52:641–652, 1998.

Use of cultured precision-cut rat lung slices to study the in vitro induction of pulmonary cytochrome P450 forms

1. The aim was to investigate the effects of some cytochrome P450 (CYP) enzyme inducers on CYP1A and CYP2B subfamily forms in cultured precision-cut rat lung slices. 2. Precision-cut lung slices were prepared from male Sprague-Dawley rats and cultured for 24 and/or 48 h in medium containing 0-20 micro g ml(-1) Aroclor 1254 (ARO), 0-50 micro M beta-naphthoflavone (BNF) and 0-50 micro M benzo(a)pyrene (BP). 3. Treatment with ARO, BNF and BP produced significant increases in lung slice whole homogenate 7-ethoxyresorufin O-deethylase activity. 4. Levels of CYP1A1 apoprotein were markedly increased in lung slice microsomes after treatment for 48 h with either 10 micro g ml(-1) ARO or 5 micro M BNF. In contrast, neither ARO nor BNF had any marked effect on levels of CYP2B1/2 apoprotein in 48-h cultured rat lung slice microsomes. 5. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan) was used to quantify lung slice CYP1A1 and CYP2B1/2 mRNA levels. Rat lung slice CYP1A1 mRNA levels were increased up to 8.3-fold after treatment for 24 h with 2 and 10 micro g ml(-1) ARO, 0.5 and 5 micro M BNF, and 20 micro M BP. In contrast, treatment with 10 micro g ml(-1) ARO produced only a small 1.6-fold increase in CYP2B1/2 mRNA levels. 6. Precision-cut lung slices are a useful model in vitro system for the assessment of the effects of chemicals on pulmonary CYP forms.

Interleukin‐10 is an Unequivocal Th2 Parameter in the Rat, whereas Interleukin‐4 is Not *

Exposure of Wistar rats to the immunotoxic compounds hexachlorobenzene (HCB), bis(tri‐n‐butyltin)oxide, and benzo(a)pyrene was previously found to affect mRNA expression of interleukin (IL)‐2, IL‐2R α‐chain, and interferon (IFN)‐γ, the prototypic Th1 cytokine. In contrast, the mRNA expression of IL‐4, the prototypic Th2 cytokine, was unaffected. This latter finding suggested that the IL‐4 mRNA expression may not be an unequivocal parameter for Th2 responses in the rat. In order to obtain such a parameter the present study was performed, consisting of two types of experiments. Expression and production of IL‐4 as well as IL‐10, a second Th2 cytokine, were measured. First, Lewis (Th1 prone) and Brown Norway (BN; Th2 prone) rats were exposed to HCB. Exposure was previously found to increase the serum immunoglobulin (Ig)E levels, an IL‐4‐dependent response, in BN but not Lewis rats, and in Lewis rats to aggravate experimental allergic encephalomyelitis (EAE), severity being inversely related to IL‐10 levels. Secondly, BN rats were infected with Trichinella spiralis, an infection previously found to induce IL‐4 production. HCB exposure did not affect IL‐4 mRNA expression in either strain, while IL‐4 production was decreased in Lewis and unaffected in BN rats. In Lewis rats both the mRNA expression and the production of IL‐10 were decreased. The T. spiralis infection induced IL‐4 and IL‐10 mRNA expression, as well as IL‐10 production. In contrast, the IL‐4 production was strongly reduced. Thus, both the IL‐10 mRNA expression and production correlated with the EAE development and T. spiralis infection. In HCB exposed Lewis rats and T. spiralis infected BN rats the IL‐4 mRNA expression correlated with IgE levels and T. spiralis infection, respectively, whereas the IL‐4 production lacked correlation in all cases. Collectively, these results suggest that IL‐10 is an unequivocal Th2 parameter in the rat, whereas IL‐4 is not.

The effect of Biostim (RU-41740) on the expression of cytokine mRNAs in murine peritoneal macrophages in vitro.

The immunomodulatory agent Biostim (RU-41740) was investigated for its ability to induce the expression of cytokine mRNAs in murine peritoneal macrophages in vitro. Northern blot analysis showed that in quiescent macrophage populations, both IL-1 alpha and IL-1 beta mRNA levels were dramatically increased in response to 1 microgram/ml Biostim. Dot-blot analysis showed that in quiescent macrophage populations the expression of mRNAs for IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha could be elevated by concentrations of Biostim as low as 1-10 pg/ml, detectable after 3 h exposure. In parallel experiments LPS was effective only at the higher concentration of 10 ng/ml. Time-course analysis showed that the expression of these cytokine mRNAs was transient, peaking after 1-3 h; only transcripts of IL-1 beta were detectable after 23 h exposure. No effects were seen on the expression of actin, a high-turnover housekeeping gene. We propose that this type of analysis represents a sensitive, specific and reproducible method for assessing the ability of drugs and chemicals to modulate the expression of cytokines that play a pivotal role in the induction of the immune response.

Transcriptome analysis provides new insights into liver changes induced in the rat upon dietary administration of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole

Transcriptomics was performed to gain insight into mechanisms of food additives butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG), and thiabendazole (TB), additives for which interactions in the liver can not be excluded. Additives were administered in diets for 28 days to Sprague-Dawley rats and cDNA microarray experiments were performed on hepatic RNA. BHT induced changes in the expression of 10 genes, including phase I (CYP2B1/2; CYP3A9; CYP2C6) and phase II metabolism (GST mu2). The CYP2B1/2 and GST expression findings were confirmed by real time RT-PCR, western blotting, and increased GST activity towards DCNB. CC altered the expression of 12 genes. Three out of these were related to peroxisomes (phytanoyl-CoA dioxygenase, enoyl-CoA hydratase; CYP4A3). Increased cyanide insensitive palmitoyl-CoA oxidation was observed, suggesting that CC is a weak peroxisome proliferator. TB changed the expression of 12 genes, including CYP1A2. In line, CYP1A2 protein expression was increased. The expression level of five genes, associated with p53 was found to change upon TB treatment, including p53 itself, GADD45alpha, DN-7, protein kinase C beta and serum albumin. These array experiments led to the novel finding that TB is capable of inducing p53 at the protein level, at least at the highest dose levels employed above the current NOAEL. The expression of eight genes changed upon PG administration. This study shows the value of gene expression profiling in food toxicology in terms of generating novel hypotheses on the mechanisms of action of food additives in relation to pathology.

Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2–200 µM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 µM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 µM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6β-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.

Sequential Effects of Dioctyltin Dichloride on the Rat Thymus

Dioctyltin dichloride (DOTC) was administered in the diet at 150 ppm to male inbred PVG rats over 2 or 4 weeks, followed by 4 weeks on normal diet. A marked reduction of thymic weight occurred (p < 0.001) and thymic weight remained reduced after reversion to normal diet. White blood counts were significantly decreased after 4 weeks treatment and remained reduced after 4 weeks recovery. In vitro lymphocyte proliferative assays demonstrated reduced mitogen-induced responses both after feeding (60%) and return to normal diet (52%) compared to untreated groups. Damage to reticular epithelium cells in the thymus, as well as lymphocyte depletion, was evident after 2 weeks treatment suggesting that DOTC can effect thymocyte maturation via thymic humoral factors.