Mary Ann Smith

An in vitro approach to the study of target organ toxicity of drugs and chemicals

SummaryA major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.

Comparative toxicity and renal distribution of the platinum analogs tetraplatin, chip and cisplatin at equimolar doses in the ficher 344 rat

Tetraplatin [tetrachloro(dl-trans)1,2-diaminocyclohexane platinum(IV), NSC-363812] is a new anticancer platinum drug analog targeted for clinical development because of its effectiveness against cisplatin-resistant tumor cell lines and its improved formulation. The toxicity of tetraplatin was compared at equimolar doses to that of cisplatin [cis-diamminedichlorophatinum(II)] and CHIP [cis-dichloro,trans-dihydroxybis-isopropylamine platinum(IV), NSC-256927]. Adult male Fischer 344 rats received an iv bolus injection of 6.7, 13.3, 26.7, or 53.3 mumol/kg of one of these drugs in saline and were killed on Day 1, 3, 5, 8, or 15 postinjection for assessment of toxicity with emphasis on evaluation of nephrotoxicity. Rats to be killed on Day 15 were housed in metabolism cages for daily urine collection. Tetraplatin was less nephrotoxic than cisplatin at equimolar doses; CHIP was not nephrotoxic at these doses. Renal platinum contents were similar after all three drugs and did not appear to be related directly to the nephrotoxicity. Nephrotoxicity was detected 4-5 days after 6.7 mumol/kg cisplatin, was localized to the corticomedullary junction, and progressed with time and dose. Tetraplatin-induced alterations of renal function were first observed after 13.4 mumol/kg on Day 4 as an elevation of urine volume (up to 10-fold) and a smaller elevation of urinary glucose excretion. Tetraplatin lesions were localized in the mid- and outer cortex and, even at the highest dose, were less severe than those observed with cisplatin. There were other prominent toxic effects of tetraplatin, such as gastrointestinal toxicity and myelosuppression, which indicate that factors other than comparative nephrotoxicity may impact the clinical potential of this new agent.

Development of a primary culture system of rat kidney cortical cells to evaluate the nephrotoxicity of xenobiotics

A method has been developed for preparing primary monolayer cultures of postnatal rat kidney cortical epithelial cells. These cultures maintained differentiated cell functions and epithelial-like morphology for several days in culture. The presence of alkaline phosphatase and maltase was used to confirm the presence of cells from the renal cortex. The concentrations of these enzymes were maintained in culture until day 3, but had declined significantly by day 5. Similar patterns were observed with cytochrome P-450 and glutathione content, although their concentrations remained stable from day 3 to day 5. Mercuric chloride, cadmium chloride and acetaminophen were evaluated for nephrotoxicity in this culture system. Treatment with these compounds resulted in dose-dependent changes in cell morphology and in biochemical parameters such as lactate dehydrogenase leakage, alkaline phosphatase activity and cellular glutathione content. With this culture system, it was possible to detect the acute toxicities of compounds that produce varying degrees of renal injury. Further development of this kidney culture system may have value in detecting potential nephrotoxins and in studying their mechanisms of toxicity.

Antibiotics taken for other illnesses and spontaneous clearance of Helicobacter pylori infection in children

Factors that determine persistence of untreated Helicobacter pylori (H. pylori) infection in childhood are not well understood. We estimated risk differences for the effect of incidental antibiotic exposure on the probability of a detected clearance at the next test after an initial detected H. pylori infection.

Cocaine-induced cardiotoxicity in vitro.

A growing number of reports have related cocaine use with the onset of myocardial infarction in young otherwise healthy individuals. Although the cardiac effects of cocaine have traditionally been attributed to sympathomimetic stimulation, several studies have suggested that cocaine may be directly cardiotoxic. The purpose of this study was to evaluate the cardiotoxic effects of cocaine in an in vitro preparation devoid of sympathetic innervation. Primary cultures of rat cardiac muscle and non-muscle cells were prepared from hearts excised from 3-5-day-old Sprague-Dawley rats. Cultures were exposed to various cocaine concentrations (1 x 10(-7)-1 x 10(-3)m) for 1-24 hr. Beating activity, morphological status and lactate dehydrogenase (LDH) leakage were evaluated following cocaine exposure. A decrease in the beating activity of cultured muscle cells was observed 1 hr after exposure to the highest cocaine concentrations (1 x 10(-5)-1 x 10(-3)m) tested. Similar results were obtained 24 hr after exposure. Morphological alterations in muscle cells were evident only after exposure to the highest concentration (1 x 10(-3)m). Vacuoles appeared 1 hr after cocaine exposure and were replaced by dark granules within 24 hr. LDH release was significantly elevated in the muscle cell cultures exposed to 1 x 10(-3)m cocaine for 24 hr. The pattern of cocaine-induced morphological alterations and enzyme leakage was similar in non-muscle cells. These data suggest that cocaine induces direct toxic effects on both cardiac muscle and non-muscle cells maintained in an environment free of neuronal and hormonal influences.

Cephaloridine toxicity in primary cultures of rat renal cortical epithelial cells.

Primary cultures of rat renal cortical epithelial cells were used to assess the in vitro nephrotoxicity of cephaloridine (Cph). Several different indices were used to follow the course of Cph-induced nephrotoxicity in the cultures. Plasma membrane integrity was determined by the effect of Cph on lactate dehydrogenase (LDH) leakage, cellular K(+) content and plasma membrane Na(+)/K(+) ATPase activity. Significant LDH leakage and decreased cellular K(+) content occurred after 8 hr of exposure to Cph. Na(+)/K(+) ATPase activity was depressed as early as 4 hr after Cph treatment. Brush border integrity was assessed by the effect of Cph on the activity of the brush border enzyme alkaline phosphatase, which was significantly decreased following 12 hr of exposure to Cph. Treatment with Cph resulted in an initial elevation of cellular glutathione (GSH)-as indicated by cellular non-protein sulphydryl content-followed by a decrease in GSH content 16 hr later. The mitochondrial response to Cph was assessed by determining mitochondrial succinate dehydrogenase (SDH) activity and cellular ATP content. SDH activity was significantly depressed after 4 hr of Cph exposure; ATP content was not significantly depressed until 12 hr after treatment. The time course of Cph-induced injury in our culture system suggests that early injury involves alterations at the level of the mitochondrial membrane and the plasma membrane. The most sensitive indicators of Cph-induced toxicity in this system were mitochondrial SDH activity and plasma membrane Na(+)/K(+) ATPase activity.

Ethanol toxicity in primary cultures of rat myocardial cells.

The potential cardiotoxicity of ethanol (EtOH) was evaluated in primary cultures of rat myocardial cells. EtOH cardiotoxicity was assessed in the cells on the basis of cell morphology, lactate dehydrogenase (LDH) leakage, succinate dehydrogenase (SDH) activity, and beating rates. Cells were treated with EtOH at concentrations of 600, 800, and 1000 mg% for duration of 1, 4, and 24 h and then evaluated for cardiotoxicity. Vacuole formation occurred 1 h after exposure to EtOH at 800 and 1000 mg%; by 4 h, cytosolic granular material appeared in these cells. Exposure for 24 h to all concentrations of EtOH resulted in vacuole, granule, and pseudopod formation and loss of cross-striations. Significant LDH leakage occurred at 1 h and 4 h with 800 and 1000 mg% EtOH. LDH release was significantly increased after 24 h with all concentrations. SDH activity was significantly depressed after 24 h with all concentrations of EtOH. Beating rates were altered as early as 1 h after exposure to 800 and 1000 mg% EtOH. After 24 h, those cells exposed to the highest concentrations of EtOH were not beating at all. These data suggest that primary myocardial cell cultures may be used to assess the in vitro cardiotoxicity of EtOH to the myocardial cell.

Lindane-Induced Generation of Reactive Oxygen Species and Depletion of Glutathione do not Result in Necrosis in Renal Distal Tubule Cells

Lindane is a chlorinated hydrocarbon pesticide, currently used in prescription shampoos and lotions to treat scabies and lice infestations. Lindane is known to be nephrotoxic; however, the mechanism of action is not well understood. In other organ systems, lindane produces cellular damage by generation of free radicals and oxidative stress. Morphological changes were observed in lindane-treated Madin–Darby canine kidney (MDCK) cells indicative of apoptosis. Lindane treatment induced time-dependent reactive oxygen species (ROS) generation. Onset of ROS generation correlated with an initial increase in total glutathione (GSH) levels above control values, with a subsequent decline in a time-dependent manner. This decline may be attributed to quenching of free radicals by GSH, thereby decreasing the cellular stores of this antioxidant. Necrotic injury was assessed by measuring lactate dehydrogenase (LDH) leakage from the cell after lindane exposure. No significant LDH leakage was noted for all concentrations tested over time. Generation of ROS and alterations in cellular protective mechanisms did not result in necrotic injury in MDCK cells, which corresponds with our morphological findings of lindane-induced apoptotic changes as opposed to necrosis in MDCK cells. Thus, lindane exposure results in oxidative damage and alterations in antioxidant response in renal distal tubule cells, followed by cell death not attributed to necrotic injury.

CHRONIC ULTRAVIOLET EXPOSURE-INDUCED p53 GENE ALTERATIONS IN SENCAR MOUSE SKIN CARCINOGENESIS MODEL

Alterations of the tumor suppresser gene p53 have been found in ultraviolet radiation (UVR) related human skin cancers and in UVR-induced murine skin tumors. However, links between p53 gene alterations and the stages of carcinogenesis induced by UVR have not been clearly defined. We established a chronic UVR exposure-induced Sencar mouse skin carcinogenesis model to determine the frequency of p53 gene alterations in different stages of carcinogenesis, including UV-exposed skin, papillomas, squamous-cell carcinomas (SCCs), and malignant spindle-cell tumors (SCTs). A high incidence of SCCs and SCTs were found in this model. Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. DNA was isolated from 40 paraffin-embedded normal skin, UV-exposed skin, and tumor sections. The p53 gene (exons 5 and 6) was amplified from the sections by using nested polymerase chain reaction (PCR). Subsequent single-strand conformation polymorphism (SSCP) assay and sequencing analysis revealed one point mutation in exon 6 (coden 193, C-->A transition) from a UV-exposed skin sample, and seven point mutations in exon 5 (codens 146, 158, 150, 165, and 161, three C-->T, two C-->A, one C-->G, and one A-->T transition, respectively) from four SCTs, two SCCs and one UV-exposed skin sample. These experimental results demonstrate that alterations in the p53 gene are frequent events in chronic UV exposure-induced SCCs and later stage SCTs in Sencar mouse skin.

Sensitivity of Urinary Enzymes as Indicators of Renal Toxicity of the Anticancer Drug Cis-platin

Rats were intravenously injected with cis-platin in order to evaluate the sensitivity of noninvasive means of detecting renal toxicity. Doses of 8 mg/kg and 2 mg/kg were used and 7 urinary parameters (osmolality, glucose, protein, and 4 enzymes) were compared with blood urea nitrogen (BUN) and histology. Urinary enzymes usually were elevated by Day 2 posttreatment and in two cases by Day 1. Protein and glucose were elevated by Day 3 and demonstrated a greater quantitative change (10-12X control) than did urinary enzymes (2-3X controls). Enzymes, protein, and glucose all returned to control levels by Day 7 or 8, and most parameters were re-elevated again by Day 10 or 12. BUN was unaltered at the lowest dose and was increased to three times control by Day 3 after the highest dose. Cis-platin induced a mild nephrosis at the lowest dose and a proximal tubular necrosis at the highest dose. The lesion occurred at the corticomedullary junction. Biochemical changes did not correspond to the times of greatest morphological changes. Large day-day and animal-animal variation made selection of a most sensitive parameter difficult. It is concluded that one parameter is insufficient to define early renal toxicity and that a battery of several parameters would provide a better evaluation of the onset of renal toxicity.