Human Proximal Tubular Cells as an In Vitro Model- for Drug Screening and Mechanistic Toxicology
Published: May 22, 2012
Dr. Lash’s research interests are in the areas of renal drug metabolism and toxicology, with particular focus on glutathione and in vitro models for study of chemically induced toxicity. Dr. Lash’s laboratory has provided a biochemical mechanism to explain the transport of glutathione into renal mitochondria, by identifying and expressing two anion carrier proteins. His laboratory has made important contributions to our understanding of the metabolism of trichloroethylene and perchloroethylene, two industrial solvents that have been identified as environmental toxins implicated in diseases including cancer. He has established in vitro cell models using rat and human kidney for use in metabolic, dispositional and toxicological studies of these solvents along with other renal toxins.
This research has resulted in 112 peer-reviewed publications and 59 reviews and book chapters and has been supported since 1986 by the National Institutes of Health, Department of Defense, U.S. Environmental Protection Agency, and the pharmaceutical industry. Dr. Lash has edited or co-edited four books on various aspects of drug metabolism and toxicology and has consulted with the National Research Council on biomarkers in urinary toxicology and with the U.S. Environmental Protection Agency on their human health risk assessments for trichloroethylene and perchloroethylene. Dr. Lash is also an Associate Editor for Toxicology and Applied Pharmacology, the Journal of Pharmacology and Experimental Therapeutics, and Pharmacology and Therapeutics and serves on the editorial boards for Toxicology, Drug Metabolism and Disposition, Toxicological Sciences, the Journal of Applied Toxicology, Chemico-Biological Interactions, and Nephroprevention. Dr. Lash was also a member of the National Institutes of Health Alcohol and Toxicology-4 Study Section (1999-2003), is currently a member of the NIDDK fellowship study section, and reviews for the NIH on an ad hoc basis for several study sections each year. He teaches medical and graduate students in the areas of drug metabolism, toxicology, pharmacogenetics, and membrane transport physiology, and has trained 4 Ph.D. and 2 M.S. students. Dr. Lash is a member of the American Association for the Advancement of Science, the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the American Society of Nephrology, the Society of Toxicology, and the International Society for the Study of Xenobiotics.
Lawrence H. Lash, Ph.D.
Department of Pharmacology
Wayne State University School of Medicine
540 East Canfield Avenue
Detroit, MI 48201 USA
Two important components of toxicity testing include drug screening, which is an important part of the drug development process, and mechanistic studies to understand how drugs or potentially toxic chemicals affect target organs. Experimental models are needed for mechanistic studies, which are important for understanding the underlying mechanisms of drug action to identify novel therapeutic targets and develop new therapeutic approaches. Animal models, including both in vivo and in vitro approaches, are commonly used in the first few phases of drug development, prior to testing in humans in clinical trials. For many chemicals, however, results from these preclinical studies in animals may not be clearly predictive of how they will act in humans. This is particularly true for chemicals that produce nephrotoxicity and nephrocarcinogenicity, because of species-dependent differences in renal metabolism and function. This essay will focus on the kidneys as a target organ, and will discuss the advantages of using primary cultures of human proximal tubular (hPT) cells as a primary experimental model.
Experimental Models for Renal Toxicity Testing
Experimental models for renal toxicity testing are summarized in Table 1. Commonly used models include laboratory animals, such as rats and mice for in vivo studies, and various in vitro models derived from both humans and other vertebrate animal species. Advantages of studies in mice include the availability of transgenics, including both knock-outs and knock-ins. Additionally, the creation of knock-in mice expressing genes of human origin (so-called ‘humanized mice’) have enabled investigation of the role of specific human genes in various physiological, pathological, and toxicological processes. While in vivo studies in rats or mice allow investigation of integrated physiological responses and determination of target organ specificity, there are limitations to the utility of such studies for predicting responses in humans (Pelekis & Krishnan, 1997; Steinberg & DeSesso, 1993). In vitro studies in tissues, tissue slices, cells, or cell lines derived from rats or mice can be very useful for mechanistic studies but may also have limited applicability in some cases for understanding processes in human tissues or cells, as discussed below.
We are of course most interested in understanding processes in humans to treat human disease. Studies of drug effects and drug actions in humans or human tissue are limited by ethical considerations or the availability of human tissue. Examples of studies in humans include epidemiological studies of human populations exposed to chemicals either in occupational settings or as a consequence of environmental contamination. While such studies have provided much useful information, there are limits to the extent and type of information that can be gained. For example, exposure conditions can only be either minimally controlled (e.g., Phase III clinical drug trial) or is out of control of the investigator and may often be known only to a limited extent (e.g., environmental or some occupational population exposures in which complete or precise exposure information is not known). Except in cases where tissue biopsies are done for diagnostic purposes, access in human subjects is typically limited to blood and/or urine samples. In some cases, human tissue that is not used for purposes of transplants can be made available for research. While such human tissue may be reasonably available for some organs, it is quite scarce for others. As will be discussed in this essay, fresh human kidneys are often reasonably available for use in research because the criteria for kidneys to be used in transplants are quite stringent. In contrast, human livers are not as readily available.
Because of these limitations, investigators often turn to various laboratory animal models, most commonly rodents such as rats and mice. Animal models run the gamut from in vivo exposures, where whole organ and integrated physiological responses are monitored, to screening or mechanistic studies involving in vitro models such as freshly isolated cells, tissue homogenates, or primary cell cultures derived from specific tissues. Such in vitro models require a continual supply of animals from which to obtain the tissue of interest. While manipulation or definition of exposure conditions is quite precise, the major limitation in their use concerns extrapolation of data to humans. In many cases, accurate extrapolation can be readily accomplished. In other cases, however, differences in responses or sensitivity between humans and other animal species are made with a considerable degree of uncertainty.
Reasons why extrapolation of animal data to humans requires care and specific information include not only quantitative differences in processes such as metabolic rate or physiological processes, such as those involving respiratory or cardiac function, but also qualitative differences. An additional complicating factor for extrapolation of data to humans concerns the existence of genetic polymorphisms, which can result in interindividual variations in activities of enzymes and membrane transporters that range from several-fold to more than one hundred-fold, due to the existence of null variants for some genes.
Extrapolation is often done by use of physiologically-based pharmacokinetic (PBPK) models and application of conversion factors to account for species differences in metabolic rate and body surface area to volume ratio (so-called allometric scaling), and then uncertainty factors to account for problems such as interindividual variation due to both genetic polymorphisms as well as dietary, social, and other environmental differences that one does not encounter in studies in rats or mice. Although incorporation of such conversion and uncertainty factors has provided the basis for many successful estimates for humans, the fact that these are best approximations still reduces the precision with which predictions can be made.
Some investigators have also used cells from non-human primates, with the assumption that they would provide data that are more readily extrapolated to humans than data derived from rodents or other vertebrate species. Caution, however, should also be exercised in the use of such data. For example, our study comparing the cytotoxicity of metalloproteinase inhibitors in primary cultures of PT cells from humans and cynomulgus monkeys (Cai et al., 2009) illustrates how species-dependent differences in responses may still exist between different primate species. Significant differences were still observed in the patterns of cytotoxicity of the various inhibitors between monkey and human PT cells, although these differences were not as great as those between rat and human PT cells.
This essay will focus on the use of human kidneys in studies of screening for nephrotoxicity and mechanism of action of drugs targeting the kidneys.
Availability of Human Kidneys
The utility of experimental models using fresh human kidneys is obviously dependent on the availability of such tissue for researchers. Although the need for kidneys for transplant is increasing due to the increasing incidence of chronic kidney disease and end-stage renal failure in the U.S. population (Atlas of Chronic Kidney Disease in the United States, 2010), many kidneys cannot be used for transplant because of the strict criteria that exist for them to be functional in the recipient. Thus, kidneys that exhibit excessive glomerulosclerosis or arterial plaque, for example, are not likely to function well when transplanted and will not be used for transplant. If the donors have consented for their organs to be used for either transplant or research, then such kidneys will become available for research.
As an example of the availability and use of fresh human kidneys for research, the following describes our experience in obtaining such organs over a period of a little more than 7 years. From January, 2001 through March, 2008 we received through Material Transfer Agreements a total of 119 human kidneys, most recently from the International Institute for the Advancement of Medicine (IIAM, Edison, NJ; http://www.iiam.org). Donor demographics were: 58 males (mean age ± SEM = 57.5 ± 1.9; range = 30-81) and 61 females (mean age ± SEM = 60.4 ± 1.3; range = 34-80). Of the 119 kidney donors, 10 were African Americans, 2 were Asians, 99 were Caucasians, 6 were Hispanics, 1 was Native American, and 1 was a Pacific Islander. Kidneys were perfused with Wisconsin or similar medium, kept on wet ice, and delivered to the laboratory within 15 to 24 hr of being taken from the donor. Kidneys were certified as normal (i.e., non-neoplastic) by a pathologist and were rejected for transplant for reasons (e.g., excessive glomerular sclerosis, arterial plaque) that do not affect our ability to isolate viable proximal tubular (PT) cells.
Limitations in the number of accepted kidneys over the above mentioned time period were due to both cost and need. Because an average human kidney yields 500 x 106 to as much as 1 x 109 PT cells, it is typically more than sufficient for most sets of experiments, limiting the need for additional tissue. With regard to expense, although each kidney costs $1,000 to $1,200 (for purchase and delivery), this is not out of line considering that the material is from humans and provides 10- to 20-fold more cellular material than a typical pair of kidneys from a rat.
Methods for PT cell isolation
hPT cells are isolated from whole human kidneys by mincing renal cortical tissue, digestion with collagenase type I and hyaluronidase, filtration through nylon sieves, and use of sterile conditions (Todd et al., 1995; Cummings & Lash, 2000; Cummings et al., 2000). All buffers are continuously bubbled with 95% O2/5% CO2 and are maintained at 37°C. Kidney cortex is sliced, washed with sterile PBS, minced, and the pieces are placed in a trypsinization flask filled with 40 ml of sterile, filtered Hanks’ buffer, containing 25 mM NaHCO3, 25 mM HEPES, pH 7.4, 0.5 mM EGTA, 0.2% (w/v) bovine serum albumin, 50 μg/ml gentamicin, 1.3 mg/ml collagenase type I, and 0.59 mg/ml CaCl2, which was filtered prior to use. Minced cortical pieces are subjected to collagenase digestion for 60 min, after which the supernatant is filtered through a 70-μm mesh filter to remove tissue fragments, centrifuged at 150 x g for 7 min, and the pellet resuspended in 1:1 Dulbecco’s Modified Eagle’s Medium:Ham’s F12 (DMEM:F12). Yield is typically ~5 to 7 x 106 cells per 1 g of human kidney cortex. Basal medium (serum-free, hormonally-defined) is DMEM:F12 (1:1) and supplements include HEPES buffer (15 mM, pH 7.4), NaHCO3 (20 mM), antibiotics (only through Day 3 of culture; 192 IU penicillin/ml + 200 µg streptomycin sulfate/ml + 2.5 µg amphotericin B/ml), insulin (5 µg/ml = 0.87 µM), transferrin (5 µg/ml = 66 nM), sodium selenite (30 nM), hydrocortisone (100 ng/ml = 0.28 µM), epidermal growth factor (100 ng/ml = 17 nM), and 3,3′,5-triiodo-DL-thyronine (T3) (7.5 pg/ml = 111 nM) (Cummings et al., 2000; Lash et al., 2001, 2003, 2005).
While the hPT cells obtained in the manner described above are predominantly (≥ 85%) of PT origin, a small degree of contamination with other epithelial cell populations will exist. Although our laboratory has typically not performed additional purification procedures with the hPT cells, options are available to further enrich the PT cell preparation. For example, density-gradient centrifugation of the renal cortical cells on Percoll can be performed to obtain PT cells that are >95% of PT origin and distal tubular (DT) cells that have minimal contamination with cells of the PT region, as we have done with cortical cells from rat kidneys (Lash & Tokarz, 1989; Lash et al., 1995).
Although primary cultures of hPT cells have the obvious advantage of using tissue from human kidney as the source, several cautions should be noted. First, maintenance of sterility is critical to prevent contamination from bacteria. Second, use of a serum-free, hormonally-defined medium is critical to maintain epithelial cell purity; whereas the typical culture medium used for established cell lines includes serum to optimize growth, its inclusion here would lead to overgrowth by fibroblasts. Finally, although the cell cultures are directly derived from PT cells and thus initially reflect all the biochemical and physiological properties of that cell population in vivo, it is well-documented that growth of cells in culture, particularly epithelial cells, can lead to a gradual or sometimes abrupt loss of certain differentiated properties. Loss of differentiated properties may include decreased expression and/or functionality of plasma membrane transporters, decreased expression of drug metabolism enzymes, loss of brush-border plasma membrane, and decreased mitochondrial function leading to cellular energy metabolism becoming primarily glycolytic rather than oxidative phosphorylation. Hence, it is critical to validate that key properties and functions of the hPT cell as they exist in vivo are maintained in primary culture.
Validation of hPT Cell Primary Cultures
An obvious prerequisite for any experimental model is that it responds to perturbations and stresses in an expected manner and exhibits expected properties and functions. Table 2 summarizes several critical properties of PT cells that should be confirmed, to consider hPT cells as a proper experimental model for drug screening, toxicity assessments, and drug development studies. These properties include morphology, drug transport and metabolism, cellular energetics, and redox status. Additional properties or functions should certainly be assessed depending on experimental needs. Examples of such properties or functions include expression of hormone receptors, synthesis of specific gene products (e.g., erythropoietin), or response to specific hormones. Thus, depending on how the cells are being used, additional evaluation criteria are required. It should be noted that genetic polymorphisms exist for several key plasma membrane transporters (Lash et al., 2006) and drug metabolism enzymes (Lash et al., 2003, 2008). Consequently, detected levels of protein expression in hPT cells of organic anion transporters (OATs), P-glycoprotein (P-gp or MDR1), cytochrome P450s (CYPs), and flavin-containing monooxygenases (FMOs), just to mention a few cases, will likely differ considerably from various donors. Hence, it is important to establish maintenance of expression levels over the course of primary culture rather than to look for specified levels of expression.
An important note about measurement of drug transport is that the hPT cells need to be grown on a matrix that enables maintenance of epithelial polarity. An example of such a matrix is Transwell filter plates. These plates contain compartments with raised, semi-permeable membranes on which the cells attach. As epithelial cells grow with their luminal or brush-border membrane facing upwards, the upper compartment of the Transwell filter plate is analogous to the tubular lumen so that addition of substrate to this compartment allows for access to the brush-border plasma membrane. In contrast, the basolateral surface faces downwards so that the lower compartment of the Transwell filter plate is analogous to the renal interstitial space and addition of substrate to this compartment allows for access to the basolateral plasma membrane.
Morphology of control or toxicant-treated hPT cells is illustrated in Figure 1. hPT cells were grown for 24 hr in the presence of either cell culture medium (= Control), 1 µM staurosporine, or 100 or 500 µM S-(1,2-dichlorovinyl)-L-cysteine (DCVC). The control cells exhibit the expected cuboidal shape with a single, large nucleus and numerous mitochondria. Staurosporine is a broad-specificity protein kinase inhibitor that causes apoptosis in many cell types (Yang et al., 1997), and has been used by us as a positive control for apoptosis in hPT cells (Lash et al., 2001). DCVC is a metabolite of the environmental contaminant and “probable human carcinogen” trichloroethylene that specifically targets mitochondria from renal PT cells and produces nephrotoxicity and renal cancer (Lash et al., 2000a, 2000b; Xu et al., 2008). DCVC is derived by conjugation of trichloroethylene with glutathione and subsequent metabolism, and can cause death of hPT cells by both necrosis and apoptosis, depending on exposure time and concentration (Lash et al., 2001). As can be seen from the photomicrographs of hPT cells exposed to either staurosporine or DCVC (Figure 1B-1D), considerable damage to cells in terms of overall shape and size and the appearance of intracellular vesicles and apoptotic bodies can be readily observed (see arrows in panels B-D).
Processes Studied in hPT Cells
A large variety of biochemical and physiological processes as well as cellular responses to various stresses can be studied in primary cultures of hPT cells, making the model very adaptable to most experimental needs. These include basic biochemical processes such as drug and intermediary metabolism, membrane transport, various assessments of mitochondrial function, various measurements of redox status and oxidative stress, assays of acute cytotoxicity, more complex and integrated processes such as protein and DNA synthesis, signal transduction pathways, assessments of gene regulation, and assays of chronic cytotoxicity, including cell death, proliferation, and repair.
Figure 2 outlines the basic types of responses of hPT cells to chemical perturbation or injury. The partitioning among the different responses will certainly differ for different classes of drugs, but is generally dependent on both exposure dose and time. For example, in a study on DCVC-induced toxicity (Lash et al., 2001), we showed that exposure of hPT cells to relatively high concentrations of DCVC (≥ 250 µM) for periods of time up to 8 hr typically results in necrotic cell death, indicative of acute tubular necrosis. In contrast, exposure of hPT cells to relatively low concentrations of DCVC (≤ 100 µM) for longer periods of time (24 to 48 hr) primarily results in apoptosis but also some evidence for enhanced cell proliferation and repair.
Figure 3 details the types of measurements that can be made to describe the cellular responses outlined in Figure 2. This scheme highlights some of the types of detailed, mechanistic studies that can be conducted when studying drug-induced nephrotoxicity. Many drugs and chemicals that produce nephrotoxicity must first be metabolized to reactive intermediates that either directly damage critical cellular molecules, such as DNA or protein, or indirectly generate reactive oxygen species (ROS), causing oxidative damage to the cell. If DNA structure or replication is altered, exposures to drugs can lead to mutations, altered gene expression, and possible genetic transformation of affected cells. Each step of these responses can readily be investigated in hPT cells.
hPT cells will grow to confluence in 5 to 7 days, depending on the growth surface and on whether or not tissue culture dishes are first coated with collagen. While it is possible to passage hPT cells to study more complex responses that require longer periods of time, caution must be exercised in using such approaches because of the previously discussed problems with de-differentiation that often occurs with extended cell culture.
Conclusions and Future Directions
An important application for the use of hPT cells that is relevant for drug screening and development is the identification of drug-drug interactions (DDIs). DDIs can be based on competition for or inhibition or induction of metabolism or membrane transport. Because of species-dependent differences in these processes, examination of DDIs in rodent models may not provide information that is readily applicable to humans. Another important application that cannot be readily modeled in non-human renal models is that of studying the influence of genetic polymorphisms on drug disposition and action. Such polymorphisms are known to exist for both drug metabolism enzymes such as CYPs and FMOs (Lash et al., 2008), and for membrane transporters such as the OATs and P-gp (Lash et al., 2006).
In summary, primary cultures of hPT cells represent a unique and highly relevant experimental model for the study of drug metabolism, transport, mechanism of action, and nephrotoxicity. Although there exist the usual cautions with primary cell culture models, and cell culture models in general, once technique is mastered and appropriate conditions are used to minimize potential issues with the culture process, the primary hPT cells have numerous advantages over other models for obtaining information about drug action that is directly relevant to humans.
©2012 Lawrence H. Lash\
|Rats/Mice: wild type||in vivo||Integrated physiological system; target-organ toxicity||Expensive; difficult to fully specify exposure; each animal serves as a single data point|
|Mice: Transgenic (KO, KI)||in vivo||Examine role of specific gene(s)||Expensive; difficult to generate|
|Mice: Humanized||in vivo||Human gene(s)||Expensive; difficult to generate|
|Isolated perfused kidney||in vitro||Intact tissue structure; distinguish intrarenal from extrarenal effects||Short-term viability (≤ 2 hr); Interanimal variability; incomplete definition of conditions; expensive|
|Renal slices||in vitro||Ease of preparation; drug screening, metabolism and transport easily measured||Short-term viability (≤ 2 hr); often limited access to luminal membrane; potential for poor oxygenation; multiple nephron cell types present|
|Isolated tubules, tubule fragments||in vitro||Intact tubular structure; determine tubular site of action; precise definition of conditions; several test samples with paired controls||Short-term viability (≤ 4 hr); often limited access to luminal membrane; some mixture of cell types|
|Freshly isolated tubular epithelial cells||in vitro||Ease of preparation; several test samples with paired controls; cells from specific nephron segments; drug screening||Short-term viability (≤ 4 hr); loss of plasma membrane polarization|
|Subcellular fractions||in vitro||Mechanistic studies; precise definition of conditions; several test samples with paired controls||Relationship to events in intact cell or organ may be difficult to establish|
|Primary cell cultures||in vitro||Closest to in vivo kidney; longer term viability; maintenance of cell polarity; drug metabolism, transport and screening; mechanistic studies||Difficult to maintain; requires animals or human donor for source of kidney cells; potential loss of differentiated functions in culture; limited lifetime relative to cell lines|
|Immortalized cell lines||in vitro||Precise definition of incubation conditions; immortalized; reproducible; easy to subculture and transfect; mechanistic studies||Often lacks some differentiated function of cell type of origin; often of ill-defined origin; difficult to relate to renal function in vivo|
|Property||Validated Characteristics||Assay Method(s)|
|Morphology||Cuboidal shape, single nucleus, numerous mitochondria||Phase-contrast microscopy|
|Drug transport-OATs, OATPs, OCTs, P-gp, MRPs, amino acid transporters|
|Drug metabolism-Phase 1: CYPs, FMOs-Phase 2: GSTs, UGTs, SULTs|
Abbreviations: CYP, cytochrome P450; FMO, flavin-containing monooxygenase; GSH, glutathione; GST, GSH S-transferase; MRP, multidrug resistance protein(ABCC gene family); OAT, organic anion transporter (SLC22 gene family); OATP, organic anion transporting polypeptide (SLCO gene families); OCT, organic cation transporter (SLC22 gene family); P-gp, P-glycoprotein (MDR1; ABCB1); SULT, sulfotransferase; UGT, UDP glucuronosyltransferase.
Figure 1. Morphology of hPT cells.
hPT cells were cultured for 5 days in supplemented, serum-free, hormonally-defined DMEM:F12 medium on 35-mm polystyrene dishes coated with Vitrogen until they were confluent. Cells were then incubated for 24 hr with either medium (= Control), 1 µM staurosporine (Sts), or 100 µM or 500 µM S-(1,2-dichlorovinyl)-L-cysteine (DCVC). Photomicrographs were taken at 100X magnification on a Carl-Zeiss Confocal Laser Microscope. Bar = 5 µm. Arrows in panels B-D indicate examples of cells with intracellular vesicles and/or apoptotic bodies.
Figure 2. Simplified scheme of drug-induced effects on renal proximal tubular cells.
Exposure of PT cells to a drug producing a toxic insult can result in a range of responses, including various mechanisms of cytotoxicity, repair and recovery, or altered cellular regulation and tumorigenesis.
Figure 3. Scheme of effects of a drug undergoing biotransformation to a reactive intermediate on renal proximal tubular cell function.
Exposure of PT cells to drugs that undergo biotransformation to a reactive intermediate may result in covalent modification of critical cellular macromolecules, directly producing damage to the cell, or may indirectly promote the formation of reactive oxygen species (ROS). Increases in ROS levels may then cause oxidative damage to critical cellular molecules, such as DNA, leading to mutations, changes in gene expression, and altered function.
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