Clive Meredith

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.

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.

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.

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.

Quantification of Cigarette Smoke Particle Deposition In Vitro Using a Triplicate Quartz Crystal Microbalance Exposure Chamber

There are a variety of smoke exposure systems available to the tobacco industry and respiratory toxicology research groups, each with their own way of diluting/delivering smoke to cell cultures. Thus a simple technique to measure dose in vitro needs to be utilised. Dosimetry—assessment of dose—is a key element in linking the biological effects of smoke generated by various exposure systems. Microbalance technology is presented as a dosimetry tool and a way of measuring whole smoke dose. Described here is a new tool to quantify diluted smoke particulate deposition in vitro. The triplicate quartz crystal microbalance (QCM) chamber measured real-time deposition of smoke at a range of dilutions 1 : 5–1 : 400 (smoke : air). Mass was read in triplicate by 3 identical QCMs installed into one in vitro exposure chamber, each in the location in which a cell culture would be exposed to smoke at the air-liquid interface. This resulted in quantification of deposited particulate matter in the range 0.21–28.00 μg/cm2. Results demonstrated that the QCM could discriminate mass between dilutions and was able to give information of regional deposition where cell cultures would usually be exposed within the chamber. Our aim is to use the QCM to support the preclinical (in vitro) evaluation of tobacco products.

The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays

Particulate matter (PM) collected from mainstream tobacco smoke is a test article commonly used for in vitro genotoxicity and cytotoxicity testing of combustible tobacco products. However, little published data exists concerning the stability of PM. We completed a 2 year study to quantify the effect of PM stor- age at 80 C, on the genotoxicity and cytotoxicity of PM generated from 3R4F and M4A reference ciga- rettes. The Ames test, Micronucleus assay (MNvit), Mouse Lymphoma assay (MLA) and the Neutral Red Uptake assay (NRU) were used. The majority of M4A and 3R4F PMs were genotoxic and cytotoxic at the timepoints tested. Some minor but statistically significant differences were observed for stored ver- sus freshly prepared PM, but the magnitude of changes were within the variability observed for repeat testing.

The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter

In vitro genotoxicity assays are often used to compare tobacco smoke particulate matter (PM) from dif- ferent cigarettes. The quantitative aspect of the comparisons requires appropriate statistical methods and replication levels, to support the interpretation in terms of power and significance. This paper recom- mends a uniform statistical analysis for the Ames test, mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT); involving a hierarchical decision process with respect to slope, fixed effect and single dose comparisons. With these methods, replication levels of 5 (Ames test TA98), 4 (Ames test TA100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) resolved a 30% difference in PM genotoxicity.

10 – Immunotoxicology Testing In Vitro

This chapter presents an overview of immunology and immune system cells and their functions, and describes the use of in vitro methods in immunotoxicology testing. The immune system is a highly complex entity involving a large number of cell types from varying lineages and is dependent for its normal function on subtle interactions between these cell types requiring both cell-cell contact and communication via soluble mediators and their receptors. Thus the whole immune system does not readily lend itself to modeling with in vitro techniques. Even though there have been great advances in cell culture technologies in recent years, with better understanding of the demands of cells for essential cytokines or growth factors for normal functioning, it is difficult to envisage any in vitro system that could mimic several of the properties of the whole immune system in vivo . Therefore, to make any progress in this field, it is necessary to break down immune function into a series of compartments, often related to known pharmacotoxicological effects of drugs and chemicals on specialized immune responses, from which can be developed appropriate in vitro systems to model specific facets of the immune system. In terms of immunotoxicology in vitro , there are many systems employed using isolated lymphocytes or monocytes which can be used in the process of drug development to assist in screening candidate molecules for efficacy or for potential undesirable side-effects. There are tremendous economic benefits in identifying proper in vitro models for the assessment of all categories of immunotoxicity, which in time would lead to the establishment of test procedures compatible with regulatory requirements.

The effect of tobacco ingredients on the in vitro mutagenicity, cytotoxicity and cell transformation potential of a novel heated tobacco product

Figure 2. THP ingredient and material risk assessment approach. • Recently, tobacco-heating products (THPs) have been developed (Figure 1) to heat rather than burn tobacco. The toxicant emissions of THPs have been shown to be considerably lower than the standard University of Kentucky reference cigarette, 3R4F[1]. • BAT has established an approach (Figure 2) to assess the toxicological risk of a novel THP device and the consumable tobacco rod (Neostik) that is heated to 240°C to release nicotine, glycerol, tobacco volatiles and any added flavour compounds. • One of the key steps in the risk assessment paradigm is to compare the in vitro toxicity of the emissions from a THP relative to that of 3R4F. • This reports the study conducted to compare the in vitro toxicity of both an unflavoured Neostik and a Neostik containing typical tobacco flavourings[2] to that of 3R4F.

Generation of in vitro margin of exposure (MOE) values to support the postulated mode of action (MOA) for selected tobacco smoke toxicants

Where the in vivo MOEs generated for individual toxicants do not provide a conclusive evaluation and are split across the critical value of 10,000 (e.g. NNK and arsenic) or where the available in vivo data is unsuitable for MOE generation (e.g. hydroquinone and catechol), the use of in vitro data can provide an alternative source of information. This can then be used to focus future research direction for each individual toxicant assessed. In the cases where in vivo and in vitro MOEs are conflicting this may indicate that a further understanding of the pathways and metabolism involved is required, in addition to potential refinement of the in vitro method.