Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: a resource for investigations into drug disposition.

Carrier-mediated transporters play a critical role in xenobiotic disposition and transporter research is complicated by species differences and their selective tissue expression. The purpose of this study was to generate a comprehensive data set of xenobiotic transporter gene expression profiles in humans and the pre-clinical species mouse, rat, beagle dog and cynomolgus monkey. mRNA expression profiles of 50 genes from the ABC, SLC and SLCO transporter superfamilies were examined in 40 human tissues by microarray analyses. Transporter genes that were identified as enriched in the liver or kidney, or that were selected for their known roles in xenobiotic disposition, were then compared in 22 tissues across the five species. Finally, as clinical variability in drug response and adverse reactions may be the result of variability in transporter gene expression, variability in the expression of selected transporter genes in 75 human liver donors were examined and compared with the highly variable drug metabolizing enzyme CYP3A4.

Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4A.

Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families.

Occupational hazard evaluation of p-bromobenzyl bromide from tests for genotoxicity.

As part of an occupational hazard evaluation, p-bromobenzyl bromide (p-BBB) was evaluated for genotoxic activity in the Ames microbial mutagenicity assay, the alkaline elution assay for DNA strand breaks in rat hepatocytes and the in vitro chromosome aberration assay in Chinese hamster ovary cells. The compound produced equivocal results in the microbial mutagenicity assay but was negative in the alkaline elution assay for DNA strand breaks in rat hepatocytes. The compound produced weakly positive results in the in vitro chromosome aberration assay. There was substantial cytotoxicity in all three assays. It is concluded that p-BBB is weakly genotoxic.

IRAG working group 5. Other assays. Interagency Regulatory Alternatives Group.

As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of the art in the development of alternative (non-whole animal) eye irritation tests, academic and industrial organizations were invited to submit in vitro eye irritation data generated in their laboratories to one of several working groups for review. The assays reviewed in this report (from Working Group 5. "Other Assays") were the EYTEX assay, tissue equivalent assay, a cytotoxicity assay using three-dimensional human fibroblast constructs, the Microtox assay, and other miscellaneous assays. Each submission consisted of raw data for chemicals and products tested, a description of the methodology, and an analysis (generally by regression analysis and Pearson's correlation coefficient) for the performance of the in vitro test relative to its ability to predict individual ocular tissue scores or total ocular score. In vivo data were generated according to the scoring methods proposed by Draize. Working Group 5 evaluated the submissions and commented on the utility of the assays. The variability of the in vivo data made conclusions difficult in many situations. Most of these assays were deemed useful (within limited chemical classes) for screening purposes or for use in conjunction with other toxicological information.

A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates.

Much of the data which have been generated on in vitro alternatives to the Draize eye irritation test have dealt with compounds within a specific chemical class or product category. However, in the pharmaceutical industry, it is often necessary to evaluate materials which are not related in structure or properties. It was thus decided to evaluate a diverse series of chemicals in seven in vitro methods for estimating ocular irritation. Thirty-seven test materials were chosen to represent a broad range of pH, solubility, and in vivo irritation potential. Assays were chosen to include as many different types of end points as practical. The group of assays was composed of TOPKAT (assessing structure-activity relationships), bovine corneal opacity-permeability (BCO-P; corneal opacity/toxicity), Eytex (protein coagulation), neutral red uptake (cytotoxicity), MTT in living dermal equivalent (cytotoxicity), Microtox (cytotoxicity in bacteria), and CAMVA (inflammation/toxicity). The results of the study indicated that, in general, the cytotoxicity end points did not correlate well with the in vivo data. The BCO-P, CAMVA, and Eytex assays had the best overall concordance (88.9, 75.8, and 75.0%, respectively) with this set of compounds. Estimation of irritation potential based on structure-activity (TOPKAT) was possible for only approximately 50% of the compounds; however, the assay showed 100% sensitivity (i.e., no false negatives), but low specificity (i.e., negatives correctly identified only 54.5% of the time). These data suggest that for screening of chemicals of diverse structure and properties, the more mechanism-based assays, as opposed to general cytotoxicity assays, hold more promise and should be further evaluated.

Assessment of cytotoxicity assays as predictors of ocular irritation of pharmaceuticals.

We have evaluated the use of cytotoxicity assays in vitro as an alternative to predicting ocular irritation potential in animals. Three different measures of cytotoxicity--leucine incorporation into protein, MTT dye reduction, and neutral red uptake--were measured in a presumed target cell, corneal epithelial cells from rabbit, as well as in a nontarget cell, V79 (Chinese hamster lung fibroblasts). An IC50 value was determined for each endpoint in one or both target cells for a series of 27 commercially available compounds and 56 in-house materials from a variety of chemical classes (carbonitriles, imidazoles, substituted benzenes, aromatic acids, peptides, phenols, esters, etc.). Analysis of the data by Spearman rho rank correlation and Pearson's correlation indicated that none of the endpoint-target cell combinations used here accurately predicts in vivo irritation potential for this group of compounds. The MTT dye reduction endpoint gave the best overall correlation, regardless of target cell, but still had a correlation coefficient below -0.5. We conclude that the measurement of cytotoxicity is of limited value as an alternative assay for the classes of materials studied here.

Bovine corneal opacity and permeability test: an in vitro assay of ocular irritancy.

Most of the published in vitro tests of ocular irritancy investigate a single parameter, generally cytotoxicity, using different cell types in culture. Although good correlations with in vivo data have been reported by some investigators, many of these studies examined only limited classes of products, mainly surfactants and cosmetic ingredients. To predict the irritant potential of compounds in development and process intermediates (which include a wide variety of chemical classes with variable physical characteristics), an assay which would allow great flexibility was needed. A recently published model of corneal opacity was appropriate for this purpose and therefore investigated. The method was substantially modified and extended to study, in the same assay, two important components of irritation, i.e., opacity and permeability. For opacity alone, values obtained for 44 common chemicals showed a correlation of r = 0.73 with published in vivo data. However, compounds like sodium lauryl sulfate and some medium-chain alcohols gave false-negative results, apparently because they produced destruction of corneal epithelium. Such an effect was quantified by the measure of corneal permeability to fluorescein, and changes observed were found to be consistent with the known irritant potential of the compounds. In combination, the measurement of these two endpoints thus appeared to be sufficient to accurately predict ocular irritancy. This was further verified with 15 process intermediates. In short, the bovine corneal opacity and permeability assay allows investigation of two important components of eye irritation, in a one-day experiment, using an ocular tissue. It represents a useful approach to assess ocular irritation at least for our needs.

Malignant transformation of a preneoplastic hamster epidermal cell line by the EJ c-Ha-ras-oncogene.

We have investigated whether the activation of endogenous ras genes is associated with the immortalization or malignant transformation of primary hamster epidermal cells by chemical carcinogens. We have also asked whether transfection of a cloned c-Ha-ras oncogene (pEJ) into a nontumorigenic cell line established from hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine treatment can induce conversion to a malignant phenotype. DNA from the nontumorigenic epidermal cell line (H5-MNNG) and from two neoplastic cell lines transformed by benzo(a)pyrene was not capable of transforming NIH/3T3 cells. This result suggests that these cells do not contain an activated (mutated) ras gene. However, when H5-MNNG cells were cotransfected with pEJ and pSV2-gpt, a plasmid containing the dominant selectable marker gene Ecogpt, seven of nine clones of Ecogpt transformants formed carcinomas in nude mice and colonies in soft agar. Southern blot analysis of BamHl-digested genomic DNA from the Ecogpt-transformed clones indicated that rapid malignant transformation was associated with integration of a complete copy of the 6.6-kilobase fragment of pEJ containing the activated c-Ha-ras gene. Furthermore, DNA from the malignant clones transformed NIH/3T3 cells in a secondary transfection assay. These studies demonstrate that a mutated c-Ha-ras gene, under the transcriptional control of its normal cellular promoter, can rapidly transform a nontumorigenic epidermal cell line. This result suggests that activation of an endogenous c-ras gene can function as the final completing event in the progression of epithelial cells to the malignant phenotype. Thus, preneoplastic cell lines of both mesenchymal and epithelial origin have now been shown to be susceptible to malignant conversion by a single mutation in a c-Ha-ras proto-oncogene.

Ornithine decarboxylase activity and DNA synthesis in primary and transformed hamster epidermal cells exposed to tumor promoter.

To better understand the progression of epithelial cells from a normal to a malignant phenotype, we have compared various responses of normal, preneoplastic, and neoplastic hamster epidermal cells to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA, but not fresh serum-containing medium, induced ornithine decarboxylase (ODC) activity in primary hamster epidermal cells; the combination of TPA and medium was no more effective than was TPA alone. In four epidermal cell lines derived after treatment of cultures with carcinogen, the pattern of ODC induction at low passage levels was similar to that observed in primary cells. However, by the time the cell lines had become anchorage independent or formed tumors in appropriate hosts, the effect of TPA and fresh medium on ODC activity was synergistic. TPA did not affect the growth rate, plateau density, cellular morphology, or size of either primary epidermal cultures or continuous cell lines. Incorporation of [3H]thymidine by primary epidermal cells and some, but not all, cell lines was stimulated by addition of fresh medium but inhibited by TPA alone. In combination with fresh medium, however, TPA potentiated [3H]thymidine incorporation. We conclude that hamster epidermal cell lines acquire a characteristic synergistic induction of ODC activity by TPA and fresh medium as they progress toward malignancy. However, no consistent alteration is observed with respect to the parameters of mitogenesis examined.

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential.

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.

Benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene metabolism and DNA adduct formation in primary cultures of hamster epidermal cells.

Primary cultures of hamster epidermal cells exposed to hydrocarbon, 1 microgram/ml, rapidly metabolized [3H]benzo(a)pyrene and [14C]7,12-dimethylbenz(a)anthracene to ethyl acetate:acetone- and water-soluble metabolites. By 24 hr, only 13.6% of the organic solvent-soluble radioactivity recovered in the medium was unchanged [3H]benzo(a)pyrene, and only 5.9% was unchanged [14C]7,12-dimethylbenz(a)anthracene. With both hydrocarbons, the major water-soluble metabolites found extracellularly were conjugated with glucuronic acid; these were primarily phenolic derivatives. Metabolites cochromatographing with 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene or trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz(a)anthracene were not detectable in high-pressure liquid chromatographic profiles of organic solvent-soluble intracellular and extracellular metabolites. However, analysis of [3H]benzo(a)pyrene: and [3H]7,12-dimethylbenz(a)anthracene: DNA adducts indicated that these putative proximate carcinogenic metabolites were formed in these cells and subsequently metabolized to DNA-binding products. The results suggest that metabolic incompetence may not be an explanation for the relative resistance of the hamster to epidermal carcinogenesis by polycyclic hydrocarbons.