Role of metabolism and oxidation-reduction cycling in the cytotoxicity of antitumor quinoneimines and quinonediimines.

Quinone(di)imines are nitrogen analogues of quinones in which one or both quinone oxygens are replaced by an imino group. A series of quinone(di)imines with antitumor activity has been studied for its in vitro chemical reactivity, metabolism, acute toxicity to primary cultured rat hepatocytes, and growth-inhibitory activity with Chinese hamster ovary (CHO) cells. The quinone(di)imines exhibited a wide range of activity as substrates for metabolism by hepatic microsomal flavoenzymes. The maximum rate of quinone(di)imine metabolism was more than 7.5-fold greater than reported for metabolism of quinones. Some quinone(di)imines formed free radicals that could be detected by electron spin resonance spectroscopy. 2-Amino-1,4-naphthoquinoneimine gave a short-lived electron spin resonance signal that could be detected only under aerobic conditions. 2,3',6-Trichloroindophenol gave an electron spin resonance signal in air that was stable for 24 h. Most quinone(di)imines underwent oxidation-reduction cycling to form the superoxide anion radical, but some quinone(di)imines, although rapidly metabolized, formed little or no superoxide anion radical. Quinone(di)imines were relatively toxic to hepatocytes and CHO cells, and some quinone(di)imines were more toxic to one cell type than the other. The log 1-octanol/water partition coefficient showed an optimal value of 2.61 for toxicity against both cell types. In hepatocytes the more toxic quinone(di)imines were the most rapidly metabolized. For a subgroup of quinone(di)imines toxicity to hepatocytes and CHO cells appeared to be related to the ability to form a semiquinone(di)imine free radical. Toxicity of quinone(di)imines to hepatocytes and CHO cells was not related to superoxide anion radical formation, and toxicity to CHO cells was not affected by exclusion of oxygen during exposure of the cells to the compounds. The rate of chemical addition of quinone(di)imines to reduced glutathione did not correlate with toxicity. An understanding of the mechanisms of acute toxicity and growth-inhibitory activity of quinone(di)imines could lead to the design of more selective quinonoid antitumor agents.

Selective inhibition of phosphatidylinositol phospholipase C by cytotoxic ether lipid analogues.

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) has been shown to be a direct inhibitor of Swiss 3T3 fibroblast and BG1 ovarian adenocarcinoma cell cytosolic phosphoinositide selective phospholipase C (PIPLC) using [3H]-phosphatidylinositol-(4, 5)-bisphosphate ([3H]PIP2) as the substrate. The inhibition occurred when ET-18-OCH3 was incorporated into the [3H]PIP2 substrate micelles, with 50% inhibition (IC50) occurring at a ET-18-OCH3: [3H]PIP2 ratio of 0.04, or an assay concentration of 0.4 microM, and when ET-18-OCH3 was added directly to the incubation, with an IC50 of 9.6 microM. Lipid prepared from cells exposed to cytotoxic concentrations of ET-18-OCH3 for 18 h also inhibited PIPLC with an IC50 less than 1 microM. The noncytotoxic analogue 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine inhibited PIPLC when incorporated into the [3H]PIP2 substrate micelles, but lipid from cells grown with 5 microM 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine did not inhibit PIPLC. BG1 cells, which were more sensitive than Swiss 3T3 fibroblasts to growth inhibition by ET-18-OCH3, had a cytosolic PIPLC activity one-third that of Swiss 3T3 cells. NIH 3T3 cells exhibited the same sensitivity to growth inhibition by ET-18-OCH3 as Swiss 3T3 cells and had a similar level of PIPLC. v-sis NIH 3T3 cells were relatively resistant (greater than 3-fold) to growth inhibition by ET-18-OCH3 and had a cytosolic PIPLC activity more than twice that of the wild type cells. ET-18-OCH3 was a weak inhibitor, IC50 greater than 100 microM, of phospholipase D activity in NIH 3T3 cell membranes. In intact NIH 3T3 cells ET-18-OCH3 at cytotoxic concentrations did not inhibit phospholipase D or phosphatidylcholine-selective phospholipase C activity. The results show that the ether lipid analogues at cytotoxic concentrations are selective inhibitors of PIPLC and that the inhibition of PIPLC may be related to the growth inhibitory activity of the ether lipid analogues.

Inhibition of growth factor-dependent inositol phosphate Ca2+ signaling by antitumor ether lipid analogues.

Cytotoxic ether lipid analogues have been studied for their ability to inhibit growth factor-dependent [Ca2+]i signaling in Swiss 3T3 fibroblasts. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) inhibited 45Ca2+ uptake and inositol(1,4,5)trisphosphate-induced 45Ca2+ release in saponin permeabilized cells with concentration producing 50% inhibition values of 55 and 360 microM, respectively. When cells were exposed to ET-18-OCH3 for 18 h before permeabilization there was selective inhibition of inositol(1,4,5)trisphosphate-induced 45Ca2+ release with a concentration producing 50% inhibition value of 20 microM, but no effect on 45Ca2+ uptake, or on 45Ca2+ release by arachidonic acid. The concentration of ET-18-OCH3 with continuous exposure to inhibit cell growth 50% was 19 microM. The ether lipid analogues 1-hexadecylthio-2-ethyl-rac-glycero-3- phosphocholine and 1-S-octadecyl-2-O-methylthiopropyl-3-N,N-dimethyl-gamma-hydroxy pro pyl ammonium iodide had effects similar to those of ET-18-OCH3 but the noncytotoxic analogue 1-alkyl-2-hydroxy-sn-glycero-3- phosphocholine was without effect. Exposure of cells to 10 microM ET-18-OCH3 produced 81% inhibition of platelet-derived growth factor-stimulated inositol phosphate formation and 66% inhibition of fluoroaluminate anion-stimulated inositol phosphate formation. Addition of ET-18-OCH3 to cells in medium with 10% fetal calf serum gave a transient increase in [Ca2+]i without causing an increase in resting [Ca2+]i, while the addition of ET-18-OCH3 to cells in medium without serum gave a sustained increase in resting [Ca2+]i. Cells exposed to 5 microM ET-18-OCH3 for 18 h showed no increase in resting [Ca2+]i but there was 95% inhibition of the [Ca2+]i response to platelet-derived growth factor, 63% inhibition of the response to bradykinin, and 55% inhibition of the response to vasopressin. The block by ether lipid analogues of inositol phosphate-mediated [Ca2+]i signaling suggests a mechanism for preventing the action of growth factors that could contribute to the inhibition of cell proliferation by the agents.

Structure of the rat collagen IV promoter.

We have isolated a 1.6 kb clone from a rat genomic library which contains the bidirectional collagen IV promoter, flanked by exons coding for the alpha 1 (IV) and alpha 2 (IV) collagen chains. There are at least two transcription start sites within both the alpha 1 (IV) and alpha 2 (IV) collagen genes. Rat mesangial cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing segments of the promoter and 5' flanking region, in both the alpha 1 (IV) and alpha 2 (IV) orientations. Our results suggest that transcriptional efficiency of the bidirectional promoter is more efficient in the alpha 2 (IV) direction than in the alpha 1 (IV) direction.

Semisynthetic pyrrolizidine alkaloid N-oxide antitumor agents. Esters of heliotridine.

The C-9 and C-7 monoesters and C-7, C-9 diesters of heliotridine with (S)-(+) and (R)-(-)-2-hydroxy-2-phenylbutyric acid were prepared, converted into their N-oxides, and compared with the corresponding C-9 monoesters of retronecine in the in vivo P388 lymphocytic leukemia screen. Relative in vitro cytotoxicities of some of the free bases and their corresponding N-oxides were also measured against the A204 rhabdomyosarcoma cell line by using the soft agar colony forming assay. Stereochemistry at C-7 of the necine and at C-2' of the necine acid appears to have a significant effect on the antitumor activity in this system. In the heliotridine series, the configuration of the necic acid has a pronounced effect on the site selectivity (C-7 vs C-9) in esterification with carbodiimidazole. An explanation for this site selectivity is offered.

Quinoneimines as substrates for quinone reductase (NAD(P)H: (quinone-acceptor)oxidoreductase) and the effect of dicumarol on their cytotoxicity.

Several quinoneimines have been shown to be substrates for partly purified rat liver cytosolic quinone reductase with either NADH or NADPH as cofactor. Km and Vmax values with NADH as cofactor for N-acetyl-p-benzoquinoneimine were 54.9 microM and 278 mumol/min/mg; for 2-amino-1,4-naphthoquinoneimine, 2.8 microM and 38 mumol/min/mg; for N,N-dimethylindoaniline, 1.7 microM and 22 mumol/min/mg; and 2-acetamido-N,N-dimethylindoaniline, 0.4 microM and 9 mumol/min/mg. All the quinoneimines showed substrate inhibition at high concentrations. At 30 microM dicumarol, an inhibitor of quinone reductase, potentiated the acute toxicity of quinoneimines to cultured phenobarbital-induced rat hepatocytes by 0.7- to 2.9-fold. Dicumarol was toxic to cultured non-induced rat hepatocytes and produced little or no increase in quinoneimine toxicity. Dicumarol potentiated the toxicity of 2-methyl-1,4-naphthoquinone (menadione) to cultured non-induced, as well as phenobarbital-induced, hepatocytes. Levels of quinone reductase in both types of hepatocytes were similar. Quinoneimines exhibited strong growth inhibitory properties with Chinese hamster ovary (CHO) cells and A204 human rhabdomyosarcoma cells. Dicumarol, 0.1 mM, potentiated growth inhibition by N,N-dimethylindoaniline and 2-acetamido-N,N-dimethylindoaniline in A204 but not in CHO cells. Growth inhibition by 2-amino-1,4-naphthoquinoneimine was inhibited by dicumarol in both cell lines. Dicumarol potentiated growth inhibition by 2-methyl-1,4-naphthoquinone in A204 and CHO cells. Quinone reductase activity in A204 cells was 48% and in CHO cells 1% of the activity in cultured hepatocytes. The lack of a correlation between the effects of dicumarol on quinoneimine and quinone growth inhibition and levels of cellular quinone reductase suggests that dicumarol has effects in cells in addition to, or other than, inhibition of quinone reductase. It is concluded that quinone reductase may protect cells against quinoneimine toxicity under certain conditions, as with phenobarbital-induced hepatocytes, but does not appear to play a major role in modifying quinoneimine toxicity in non-induced hepatocytes, or growth inhibition in CHO cells or A204 cells.

Toxicity of the sulfhydryl-containing radioprotector dithiothreitol.

The toxicity of the sulfhydryl-containing radioprotective agent dithiothreitol (DTT) has been studied using Chinese hamster V79 cells growing in monolayer in minimal essential medium containing 10% fetal calf serum. DTT at low concentrations (between 0.4 and 1.0 mM) caused cell killing, but higher concentrations (above 2 mM) or lower concentrations (0.1 mM) did not. This DTT-induced toxicity was prevented by catalase, glutathione, the use of serum-free medium, or lowering incubation temperature; was slightly decreased by dimethyl sulfoxide; and was enhanced by some metal chelators but prevented by desferal, an iron chelator. Experiments involving simultaneous exposure of cells to DTT and H2O2 showed that low concentrations of DTT enhanced H2O2-induced toxicity, but high concentrations of DTT prevented the H2O2 toxicity. These results are consistent with the proposal that toxicity results from autoxidation of DTT to produce H2O2, which in turn reacts via the metal-catalyzed Fenton reaction to produce the ultimate toxin, .OH radicals, although chemical studies show that rates of autoxidation of various sulfhydryl compounds do not correlate with the observed toxicity.

Interactions of radioprotectors and oxygen in cultured mammalian cells. II. Effects of dithiothreitol on radiation-induced DNA damage and comparison with cell survival.

The effects of the sulfhydryl-containing compound dithiothreitol (DTT) on radiation-induced DNA damage have been studied using two different assays: DNA unwinding hydroxyapatite chromatography and alkaline filter elution. DNA damage as measured by both assays for cells irradiated in air shows drug concentration-dependent radioprotection reaching high levels (dose reduction factor, DRF = 3) at high DTT concentrations. The pattern and degree of protection against DNA damage are the same as shown previously for cell survival. However, when cells are irradiated in hypoxia, DNA damage as measured by the unwinding technique is decreased less by low DTT concentrations than is survival, but DNA damage is decreased to a much greater extent (DRF = 3) at high concentrations of DTT (compared to DRF = 1.5 for cell survival). DNA damage as measured by the alkaline elution assay after hypoxic irradiation is decreased to a much greater extent at all concentrations of DTT with DRF = 1.6 at 1 mM and increasing to DRF = 4.5 at high levels of DTT. These results are discussed in terms of the different types of DNA damage produced in cells irradiated in air versus hypoxia and the differences in types of damage measured by the two different DNA assays and cell survival.

Radiation-induced binding of DNA from irradiated mammalian cells to hydroxyapatite columns.

In experiments designed to measure radiation-induced DNA damage using the DNA unwinding-hydroxyapatite chromatography technique, we observed that under some experimental conditions a significant proportion of the test DNA became tightly bound to the hydroxyapatite (HA) and could not be released even with a high concentration of phosphate buffer. Approximately 5-10% of DNA from unirradiated cells binds to the HA. With increasing radiation doses in air, the fraction of bound DNA increases, reaching about 30% at about 35 Gy. The binding exhibits many of the characteristics of a radiation-induced cell lesion: the proportion of DNA retained by the HA is less when cells are irradiated under hypoxic conditions or in the presence of the thiol radioprotector dithiothreitol; and the binding decreases when an incubation period is allowed between irradiation and harvest of the cells for assay. Studies to determine the nature of the lesion responsible for the binding demonstrated that lesion production requires a component found in cells since no binding was observed with irradiated isolated DNA or nuclear matrix; the binding is not a result of the production of DNA-protein crosslinks; and the bound DNA is single-stranded, based on its sensitivity to nuclease S1. Because of the dose dependence of the binding of DNA to HA, the slopes of the dose-response curves for DNA damage determined with this assay depend on the method used to calculate the fraction of double-stranded DNA. Our demonstration that the bound DNA is single-stranded guides the choice of the method for data analysis.

Isolation, identification and biological activity of a phyllanthoside metabolite produced in vitro by mouse plasma.

The antitumor agent phyllanthoside is rapidly metabolized in vitro by mouse plasma. This metabolite has now been isolated from mouse plasma and its structural properties and cytotoxicity characterized. The isolated metabolite was estimated to be greater than 98% pure by HPLC analysis. Mass spectral analysis (fast atom bombardment and tandem mass spectrometry) indicated that the metabolite was the aglycone of phyllanthoside that resulted from the cleavage of the ester bond linking the aglycone and the disaccharide moieties of phyllanthoside. This identification was based on identical collision-induced dissociation spectra of both phyllanthoside and the metabolite. The aglycone was not formed by mouse plasma that had been boiled, filtered to remove proteins, or treated with 1.0 mM diisopropyl fluorophosphate. These results suggest that aglycone formation occurs as a result of plasma esterase activity. Michaelis-Menten constants, Vmax and Km, for conversion of phyllanthoside to the aglycone at 22 degrees C were estimated to be 1.1 mmol/ml plasma/min and 2.0 mM, respectively. Concentrations of phyllanthoside and metabolite required to inhibit cell-colony formation by human A204 rhabdomyosarcoma in vitro were 0.47 nM and 24 microM, respectively. The toxicity of phyllanthoside, and perhaps its efficacy as an antitumor agent in mice, may depend on its rate of conversion to the aglycone.

D-3-deoxy-3-substituted myo-inositol analogues as inhibitors of cell growth.

A number of unnatural D-3-deoxy-3-substituted myo-inositols were synthesized and their effects on the growth of wild-type NIH 3T3 cells and oncogene-transformed NIH 3T3 cells were studied. The compounds were found to exhibit a diversity of growth-inhibitory activities and showed selectivity in inhibiting the growth of some transformed cells as compared with wild-type cells. Remarkably, D-3-deoxy-3-azido-myo-inositol exhibited potent growth-inhibitory effects toward v-sis-transformed NIH 3T3 cells but had little effect on the growth of wild-type cells. The growth-inhibitory effects of the myo-inositol analogues were antagonized by myo-inositol. Since [3H]-3-deoxy-3-fluoro-myo-inositol was shown to be taken up by cells and incorporated into cellular phospholipids, we suggest that these unnatural myo-inositol analogues may act as antimetabolites in the phosphatidylinositol intracellular signalling pathways. Because cells transformed by oncogenes often exhibit elevated phosphatidylinositol turnover, the inhibition of signalling pathways that mediate oncogene action could offer new opportunities for controlling the growth of cancer cells.

Inhibition of growth factor binding, Ca2+ signaling and cell growth by polysulfonated azo dyes related to the antitumor agent suramin.

The ability of the polysulfonated antitumor drug suramin and six related polysulfonated azo dyes to inhibit the cell growth, platelet-derived growth factor (PDGF)-receptor binding, and intracellular Ca2+ signaling of Swiss 3T3 fibroblasts was studied. Some of the azo dyes were more potent inhibitors of PDGF binding than was suramin. The concentration giving 50% inhibition (IC50) of PDGF binding was 0.5 microM for the most potent azo dye as compared with 10 microM for suramin. The azo dyes were generally more potent inhibitors of nonmitochondrial Ca2+ uptake and of inositol(1,4,5)trisphosphate-mediated Ca2+ release in permeabilized Swiss 3T3 cells than was suramin, and they were more potent inhibitors of PDGF-induced Ca2+ signaling in intact Swiss 3T3 cells. The azo dyes were only as effective as or less effective than suramin in inhibiting the growth of Swiss 3T3 cells, with IC50 values of between 74 and 361 microM being noted for the dyes as compared with 70 microM for suramin. The difference between the growth-inhibitory activity of the azo dyes and that of suramin could not be explained by metabolism of the compounds, which was not detectable in either Swiss 3T3 cells or human liver slice preparations. The results suggest that suramin and some of the azo dyes have actions on cell growth in addition to inhibition of growth factor binding and of Ca2+ signaling.

Platelet-derived growth factor blocks the increase in intracellular free Ca2+ caused by calcium ionophores and a volatile anesthetic agent in Swiss 3T3 fibroblasts without altering toxicity.

Platelet-derived growth factor (PDGF) produced an almost complete block of the increase in intracellular free Ca2+ concentration ([Ca2+]i) in Swiss 3T3 fibroblasts caused by the Ca2(+)-selective ionophores 4-bromo-A23187 and ionomycin, and by the volatile anesthetic agent halothane. The effect of PDGF was similar to the decreased [Ca2+]i response to Ca2(+)-ionophores produced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. There was no effect of PDGF or PMA on the acute or delayed toxicity of the Ca2(+)-ionophores to Swiss 3T3 cells, suggesting that the increase in [Ca2+]i is not the direct cause of toxicity of these agents.

Transforming growth factor-beta 1 induces collagen IV gene expression in NIH-3T3 cells.

BACKGROUND: While recent studies have implicated transforming growth factor-beta 1 (TGF-beta 1) in the development of glomerular scarring, extraglomerular matrix production is frequently associated with glomerulonephritis and is an important determinant of disease progression. TGF-beta 1 may be an important mediator of extracellular matrix synthesis, both by glomerular and extraglomerular mesenchymal cells. TGF-beta 1-mediated collagen IV gene expression was studied in two mesenchymal cell lines. Initial studies were performed utilizing NIH-3T3 cells, a fibroblast-like line derived from murine embryo that has been used to study regulation of fibrillar collagen (collagen I and collagen III) synthesis by TGF-beta 1. Additional studies were performed using normal rat kidney cells (NRK-49F). EXPERIMENTAL DESIGN: Cells were grown in medium supplemented with 0.5% calf serum for 24 hours before treatment with TGF-beta 1. RNA was isolated after the addition of varying amounts of TGF-beta 1 to the cells in culture for varying periods of time, and collagen alpha 1(IV) RNA was quantitated by filter hybridization. Transcription of the alpha 1(IV) and alpha 2(IV) collagen genes was assessed by an in vitro transcription assay. Deposition of collagen IV was identified by immunoblotting. RESULTS: Induction of alpha 1(IV) gene expression by NIH-3T3 cells and by NRK-49F cells was first seen 2 to 4 hours after TGF-beta 1 treatment, and was maximal after 12 to 18 hours. Maximal induction was observed following addition of 5 ng/ml TGF-beta 1 to NIH-3T3 cells, and following addition of 10 ng/ml of TGF-beta 1 to NRK-49F cells. In the presence of cycloheximide, TGF-beta 1 induction of alpha 1(IV) mRNA was markedly attenuated in both cell lines, suggesting that this effect of TGF-beta 1 requires protein synthesis. TGF-beta 1 increased transcription of both the alpha 1(IV) and alpha 2(IV) collagen genes by NIH-3T3 cells. CONCLUSIONS: TGF-beta 1 induces collagen IV gene expression in both NIH-3T3 cells and normal rat kidney fibroblasts (NRK-49F cells). Further studies of cytokine-mediated transcriptional regulation of collagen IV, utilizing these cell lines, may provide important information regarding the role of extraglomerular matrix production in the progression of renal disease.

Human and dog, but not rat, isolated hepatocytes have decreased foreign compound-metabolizing activity compared to liver slices.

A comparison has been made of the metabolism of biphenyl by isolated hepatocytes and liver slices from rat, dog, and human. Hepatocytes were prepared by low Ca2+ and enzyme digestion of the perfused liver of rat or liver slices from the rat, dog, and human. The ratio of free to total hydroxybiphenyl formation (R) was a sensitive measure of hepatocyte functional viability in perfusion-isolated rat hepatocytes, showing a significant negative correlation (r = -0.920, p less than 0.01) with trypan blue exclusion (TBE). Rs for rat hepatocytes prepared by the perfusion and slice-digestion techniques were not significantly different. Biphenyl metabolism and TBE in rat, dog, and human hepatocytes isolated by the slice-digestion technique were compared. Total hydroxybiphenyl formation by dog and human hepatocytes was 21% and 4% of that seen with rat hepatocytes. Rs for rat, dog, and human hepatocytes were 0.19, 0.46, and 0.63, respectively. TBE for all the hepatocyte preparations was approximately 90%. In contrast to the hepatocytes, total hydroxybiphenyl formation by slices of dog and human liver was 106% and 108%, respectively, of that seen with slices of rat liver. Rs for rat, dog, and human liver slices were 0.11, 0.21, and 0.26, respectively. These results suggest that hepatocytes prepared by the slice-digestion technique from dog and human but not rat liver have lost some of their ability to oxidize biphenyl and form biphenyl conjugates. This may be due to damage to the hepatocytes during isolation. TBE does not appear to be an accurate measure of hepatocyte functional viability between species. It is concluded that liver slices may provide a better model than isolated hepatocytes for foreign compound metabolism studies with dog and human liver.

Identification of key residues in subgroup A avian leukosis virus envelope determining receptor binding affinity and infectivity of cells expressing chicken or quail Tva receptor.

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.

Studies of chemical toxicity to fresh and cryopreserved rat hepatocytes.

Isolated hepatocytes are useful for studying the metabolism and mechanisms of hepatic toxicity of foreign chemicals. A problem with using human hepatocytes is the limited and irregular availability of normal human liver. Cryopreservation could provide a useful way of storing hepatocytes until they are needed. As a preliminary step to using human hepatocytes we have compared the toxic response to chemical toxicants of primary cultures of fresh rat hepatocytes and rat hepatocytes cryopreserved as previously described (G. Powis, K. S. Santone, D. C. Melder, L. Thomas, D. J. Moore, and T. J. Wilke, 1987. Drug Metab. Dispos. 15, 826). After 24 hr in culture the cryopreserved hepatocytes had a plating efficiency 75% that of noncryopreserved hepatocytes. The cultured cryopreserved hepatocytes showed a small increase in spontaneous lactate dehydrogenase release compared to that of cultured noncryopreserved hepatocytes. A similar toxic chemical-induced increase in lactate dehydrogenase release occurred in the cultured cryopreserved as in the noncryopreserved hepatocytes. The 50% effective concentrations (EC50) for lactate dehydrogenase release (+/- SE, n = 3 preparations) from cultured cryopreserved and noncryopreserved hepatocytes for chlorpromazine were 235 +/- 20 and 215 +/- 30 microM, for cadmium chloride 200 +/- 5 and 272 +/- 23 microM, and for menadione (2-methyl-1,4-naphthoquinone) 24 +/- 7 and 44 +/- 8 microM, respectively. The EC50 values for intracellular glutathione depletion in cultured cryopreserved and noncryopreserved hepatocytes were for chlorpromazine 200 +/- 8 and 235 +/- 8 microM, for cadmium chloride 242 +/- 19 and 213 +/- 7 microM, and for menadione 22 +/- 2 and 21 +/- 3 microM, respectively. The results show that cryopreservation offers a practical way of storing rat hepatocytes for studies of chemical toxicity.

Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factor-beta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV.

Transforming growth factor-beta1 (TGF-beta1) is well recognized as a potent mediator of both fibrillar (collagen type I) and basement membrane (collagen type IV) production. However, tissue injury is characterized by the concomitant expression of many cytokines and/or growth factors in addition to TGF-beta1, and the ultimate extent of extracellular-matrix (ECM) deposition may reflect the interacting effects of TGF-beta1 and these other cytokines and/or growth factors. We, therefore, sought to determine whether other cytokines and/or growth factors, known to be produced after tissue injury, are capable either alone or in combination with TGF-beta1 of modulating collagen gene expression. Collagen type I and collagen type IV gene expression was assessed in NIH-3T3 cells, a murine fibroblast-like cell line that responds to TGF-beta1, with increases in both collagen type I and collagen type IV production. TGF-beta1 coordinately induced production of collagen type IV messenger ribonucleic acid (mRNA) to a level 3.8-fold above its baseline value (p < 0.001) and collagen type I mRNA to a level 2.6-fold above its baseline value (p < 0.001). Of the other cytokines and/or growth factors tested, only epidermal growth factor (EGF) had significant effects on collagen mRNA expression. We report the novel observation that EGF significantly induced collagen type IV mRNA (3.0-fold; p < 0.001) but did not alter collagen type I mRNA expression. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and insulin-like growth factor-1 (IGF-1) did not alter the expression of mRNA for collagen type IV or collagen type I. Addition of TGF-beta1 to cytokine- and/or growth factor-treated cells increased both collagen type IV and collagen type I mRNA levels. However, collagen type IV mRNA levels were similar in cultures given TGF-beta1 alone and cultures given TGF-beta1 with other cytokines and/or growth factors; there were no additive, synergistic, or antagonistic effects after coadministration of TGF-beta1 and other cytokines and/or growth factors. With regard to collagen type I mRNA expression, all cytokines and/or growth factors tested, with the exception of TNF-alpha, had no effect on collagen type I mRNA levels in TGF-beta1-treated cultures. Importantly, TNF-alpha antagonized the stimulatory effect of TGF-beta1 on collagen type I mRNA levels. These observations support a dominant role for TGF-beta1 in stimulating coordinate expression of collagen type I and collagen type IV mRNAs by NIH-3T3 cells; EGF and TNF-alpha are capable of inducing divergent expression of the genes for these two types of collagen.

An increase in intracellular free Ca2+ associated with serum-free growth stimulation of Swiss 3T3 fibroblasts by epidermal growth factor in the presence of bradykinin.

Bradykinin gave a biphasic increase in intracellular free Ca2+ concentration ([Ca2+]i) in serum-deprived Swiss 3T3 fibroblasts loaded with the photoprotein aequorin. Epidermal growth factor (EGF) alone did not increase [Ca2+]i, but when added after bradykinin there was an increase in [Ca2+]i. The EGF-dependent increase in [Ca2+]i was maximal at 3 min and disappeared with a half-life of 6 min after bradykinin. Removing Ca2+ from the external medium did not abolish either the bradykinin or the EGF-induced [Ca2+]i responses. Although prostaglandins E2 and F2 alpha also gave [Ca2+]i responses and permitted an EGF-dependent [Ca2+]i response, the effect of bradykinin did not appear to be mediated by prostaglandins since it was not blocked by indomethacin. Vasopressin and phorbol 12-myristate 13-acetate both gave a [Ca2+]i response but did not facilitate a [Ca2+]i response by EGF. Bradykinin or EGF alone did not increase DNA synthesis in growth-arrested Swiss 3T3 fibroblasts, but EGF added together with, or after, bradykinin increased DNA synthesis. The effect disappeared with a half-life of 180 min after the addition of bradykinin. It is concluded that stimulation of receptor protein tyrosine kinase is unlikely, by itself, to explain the increase in DNA synthesis produced by EGF. The observed increase in [Ca2+]i caused by EGF after bradykinin probably reflects the interaction of intracellular second messenger pathways leading to facilitation of DNA synthesis.