Trolox Inhibits Peroxynitrite-Mediated Oxidative Stress and Apoptosis in Rat Thymocytes

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Vol. 333, No. 2, September 15, pp. 482–488, 1996 Article No. 0418

Trolox Inhibits Peroxynitrite-Mediated Oxidative Stress and Apoptosis in Rat Thymocytes Maria G. Salgo and William A. Pryor1 The Biodynamics Institute, 711 Choppin Hall, Louisiana State University, Baton Rouge, Louisiana 70803-1800

Received May 9, 1996, and in revised form July 8, 1996

Peroxynitrite is a strong oxidant that reacts with a variety of biomolecules in vivo and in vitro. When rat thymocytes in phosphate buffer are exposed to 25 mM peroxynitrite for 10 min, DNA single strand breaks (SSB) can be detected. These SSB are repaired if the cells are incubated in fresh media at 377C for 120 min. In addition, DNA protein cross-links and apoptosis are observed 1 and 6 h, respectively, after peroxynitrite exposure. Peroxynitrite mediates the formation of thiobarbituric acid-reactive substances (TBARS) that may be responsible for the DNA–protein crosslinks (DPXL). Both TBARS and DPXL formation are lowered by posttreating the cells immediately after the 10-min exposure to peroxynitrite with Trolox, a water-soluble vitamin E analog. These results suggest that oxidative stress mediated by peroxynitrite can trigger a critical sequence of events ending in programmed cell death and that intracellular oxidation is a component of the apoptosis of thymocytes, since both oxidative processes and apoptosis can be prevented by Trolox. In addition to Trolox, we obtained partial data on three other phenolic antioxidants (3-tert-butyl-4-hydroxyanisole, butylated hydroxytoluene, and 2,6-diisopropylphenol). We find that Trolox and these three phenols similarly protect rat thymocytes from apoptosis mediated by peroxynitrite. q 1996 Academic Press, Inc. Key Words: DNA damage; rat thymocytes; apoptosis; DNA–protein cross-link; free radical scavenger; peroxynitrite.

vent or minimize oxidative injury, damage does occur, presumably in situations where there is an increased activity of radical/oxidant production or a decreased level of defenses (2). An example of such a situation occurs when two radicals such as nitric oxide NO•, a moderately oxidizing agent, and superoxide O•0 2 , a reducing agent, are simultaneously produced and react to form peroxynitrite, 0OONO (3–12). Peroxynitrite is a strong, versatile oxidant that is capable of reacting with all major classes of biomolecules (8–12). Peroxynitrite is also able to cause single strand breaks in supercoiled DNA (13), in rat thymocyte DNA (14), and in macrophage and smooth muscle cell DNA (15). Peroxynitrite can induce apoptosis in a variety of cell types (16–18). Apoptosis is a programmed form of cell death (19, 20) characterized by a variety of morphological, biochemical, and genetic markers (21). Very little is known about the mechanisms by which apoptosis is triggered, but it appears increasingly likely that intracellular oxidation plays a central role in apoptotic cell death (22, 23), since many types of oxidants induce apoptosis and antioxidants and free radical scavengers inhibit apoptosis (24). Here we report several aspects of peroxynitrite-mediated cell damage using primary rat thymocyte cell cultures as our model. Thymocytes are extremely susceptible to apoptosis and are consequently one of the bestcharacterized cell systems for studying such phenomena. We have followed the sequence of events mediated by a single bolus addition of 25 mM peroxynitrite to rat thymocytes.

Oxidative stress may be defined as the state in which exposure to oxidative insult, often involving free radicals, represents a challenge to normal cellular function and even cellular survival (1). Although organisms have developed an extensive array of defenses to pre-

MATERIALS AND METHODS

1 To whom correspondence should be addressed. Fax: (504) 3884936. E-mail: [email protected].

2 Abbreviations used: FADU assay, fluorescence analysis of DNA unwinding; DPLX, DNA–protein cross-link; TBA, thiobarbituric

Materials Trolox, Hoechst 3358, and malonaldehyde bis(dimethylacetal) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sodium azide was from EM Science (Gibbston, NJ). Proteinase K, agarose, diphenylamine, phenylmethylsulfonyl fluoride (PMSF),2 polyoxyeth-

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TROLOX PROTECTS CELLS AGAINST PEROXYNITRITE ylene ether W-1, 3-t-butyl-4-hydroxyanisole (BHA), butylated hydroxytoluene (BHT), 2,6-diisopropylphenol (DIP), and calf thymus DNA were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal calf serum, RPMI 1640, antibiotic/antimycotic, and the 1-kb DNA ladder were from GibcoBRL (Gaithersburg, MD).

Methods Peroxynitrite synthesis. Peroxynitrite was synthesized by ozonation of an aqueous solution of 0.1 M sodium azide at pH 12, as described earlier (25). To minimize contamination by unreacted azide, ozonation was continued even after a maximal yield of peroxynitrite occurred (26). Our 25 mM peroxynitrite stock solution was diluted down to a working concentration of 25 mM, which contained traces (5 nM) of unreacted azide and was essentially free of hydrogen peroxide. Isolation of thymocytes. Male Sprague–Dawley specific pathogen-free rats 4–7 weeks old (Harlan Sprague–Dawley, Houston, TX) were anesthetized with phenobarbital and then sacrificed by exsanguination. The thymus was removed, placed in a saline solution kept at 377C, and mashed with tweezers to release the thymocytes. The cells were pelleted by centrifugation at 400g at 47C and resuspended in 0.87% ammonium chloride/10 mM Tris–HCl, pH 7.2, 10 mM sodium bicarbonate solution to lyse any red blood cells. The thymocytes were centrifuged again and the pellet was resuspended in 100 mM phosphate buffer, pH 7.4, containing 0.1 mM DTPA. The thymocytes were counted, and their viability was determined by the trypan blue exclusion method. Typically, viability was about 98% and 1.5–1.8 1 109 cells were harvested from each gland. Treatment with peroxynitrite. Thymocytes (16 million cells/tube) were resuspended in 100 mM phosphate buffer, pH 7.4, containing 0.1 mM DTPA and treated with a single bolus addition of peroxynitrite (25 mM final concentration) at room temperature. Decomposed peroxynitrite was prepared by diluting an aliquot of the stock solution in water, adjusting the pH to 2, and allowing the peroxynitrite to decompose at room temperature for 30 min. Incubation of thymocytes after peroxynitrite treatment. After peroxynitrite or decomposed peroxynitrite treatment, the cells were pelleted by brief centrifugation, resuspended in tissue culture media, and plated in 35 1 10-mm petri dishes to give 5 1 106 cells/ml. Tissue culture media were prepared using RPMI 1640 supplemented with L-glutamine, 1% antibiotic/antimitotic, and 10% fetal bovine serum. For the experiment involving Trolox, the antioxidant was first dissolved in media (RPMI 1640), a few drops of sodium bicarbonate were added to increase its solubility, and then the solution was added to the plate to give a final concentration of Trolox of 10 mM. For the experiments involving BHA, BHT, and DIP, the antioxidants were dissolved in ethanol and added to the plate to give a final concentration of 5 mM (0.5% of the media volume). The control cells for this experiment were incubated in media containing 0.5% ethanol. This set of treated cells was assessed for apoptosis by gel electrophoresis analysis. Incubation was carried out at 377C under an atmosphere of 5% CO2 and 95% oxygen for 10 min up to 6 h. We then measured DNA single strand breaks and their repair, DNA–protein crosslinks, thiobarbituric acid-reactive substances (TBARS), DNA fragmentation, and apoptosis. Determination of DNA single strand breaks and repair. The formation of DNA strand breaks was determined using the fluorescence analysis of a DNA unwinding assay (FADU)(27). After treatment with peroxynitrite and a subsequent incubation in fresh media, the cells were harvested at 10, 20, 40, 60, 90, and 120 min, then pelleted by centrifugation and resuspended in buffered isotonic solution, pH

acid; TBARS, thiobarbituric acid-reactive substances; BHA, 3-t-butyl-4-hydroxyanisole; BHT, butylated hydroxytoluene; DIP, 2,6-diisopropylphenol; PMSF, phenylmethylsulfonyl fluoride; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate-buffered saline.

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7.2, containing 0.25 M myoinositol, 10 mM sodium phosphate, 1 mM magnesium chloride, and processed for the FADU assay. Briefly, the cells were lysed with a urea/detergent solution, alkali was added, and the crude extracts were incubated for 30 min on ice and then for another 45 min at 157C. After neutralization, ethidium bromide was added and the fluorescence measurements were taken. Detection of DNA–protein cross-links (DPXL). Isolated thymocytes were first treated with peroxynitrite and plated as described above. After the incubation the thymocytes were processed for detection of DPXL; the assay was carried out according the method of Zhitkovich and Costa using SDS-K/ precipitation (28). Aliquots (100 ml) containing 2 million cells were transferred to Eppendorf tubes and lysed with 0.5 ml of 2% SDS, 1 mM PMSF, and 20 mM Tris– HCl (pH 7.5). The samples were kept at 0607C overnight. After thawing, the mixture was vortexed and warmed for 10 min at 657C, and then 0.5 ml of 200 mM KCl in Tris–HCl, pH 7.5, was added. The mixture was passed through a 1-ml pipet six times to shear the DNA. The samples were then cooled on ice and centrifuged at 3000g for 5 min at 47C. Supernatant solution containing unbound DNA was collected. Protein precipitates, which contained DPXL, were washed three times by resuspension in 0.1 M KCl, 0.1 mM EDTA, 10 mM Tris. Precipitates were collected by centrifugation as described earlier, and supernatant solutions containing the unbound DNA fraction from each wash were combined. The final pellet was resuspended into 1 ml of washing buffer and 0.2 mg/sample of proteinase K was added. The samples were kept at 507C for 4 h. The digests were placed on ice for 5 min and centrifuged at 10,000g for 10 min. The DNA content in each sample was determined using the Hoechst reagent (29, 30). The supernatant solutions containing the unbound DNA as well the supernatant from the final digestion were mixed with 1 ml of freshly made Hoechst reagent (250 ng/ml). The samples were incubated for 30 min in the dark. Fluorescent measurements were taken using a Perkin–Elmer LS 50 luminescence spectrometer (Perkin–Elmer, Norwalk, CT) using excitation at 360 and emission at 450 nm. To measure the DNA content in each sample, a standard curve was prepared using calf thymus DNA, containing 0, 1.25, 2.5, 5, 10, or 20 mg/ml. Fluorescence enhancement averaged about 10 units per microgram of DNA. Determination of TBARS. As a measure of lipid autooxidation products, we used the thiobarbituric acid (TBA) test. After peroxynitrite treatment and incubation in media for different lengths of time, the cells were pelleted and resuspended in a small volume of PBS (100 ml). The suspension was mixed with 100 ml of a 10% solution of polyoxyethylene ether W-1 (nonionic detergent), 250 ml of 40% trichloroacetic acid (TCA), and 600 ml of an aqueous solution containing 0.75% TBA. The samples were heated in boiling water for 30 min, cooled, and centrifuged, and the absorbance was measured at 532 nm. Yields of malonaldehyde were calculated using malonaldehyde diacetal as a standard (31). DNA extraction and electrophoresis. Thymocytes were treated with peroxynitrite, incubated in media as above, with or without antioxidants, and harvested by centrifugation for 30, 60, 120, 240, or 360 min. The pellets were lysed with 0.2 ml of lysis buffer (10 mM Tris–HCl, pH 7.5/1 mM EDTA/0.2% Triton X-100), 50 mg/ml of proteinase K was added, and the samples were incubated for 1 h at 377C. The DNA was extracted by adding an equal volume of phenol/ chloroform/isoamyl alcohol (25:24:1), and the recovered DNA was precipitated by adding 2 vol of 100% cold ethanol and allowed to precipitate overnight at 0207C. The DNA was then collected by centrifugation, air dried, and resuspended in 15 ml of 10 mM Tris/1 mM EDTA buffer, pH 8. The samples were mixed with 5 ml of gel loading buffer [0.33% bromophenol blue and 40% (w/v) sucrose] and loaded onto a 0.8% agarose gel. Ethidium bromide was included to allow visualization of the DNA under uv light. DNA fragmentation and quantitation assay. After treatment with peroxynitrite, the cells were harvested, resuspended in tissue culture media, and incubated for the indicated time (30, 60, 120, 240,

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FIG. 1. Effect of exposure to peroxynitrite (h) and previously decomposed peroxynitrite (l) on rat thymocyte viability. Control cells (j) and peroxynitrite-treated cells (s) were incubated in media fresh containing 10 mM Trolox. After exposure, the cells were incubated in media at 377C and viability was measured after 30, 60, 120, 240, or 360 min. Trolox was added to the unexposed cells as well (n).

Cells treated with decomposed peroxynitrite show a drop in viability corresponding to 50% of that caused by 25 mM peroxynitrite. Peroxynitrite and its decomposition products have an effect on cell viability. The observation that the viability of the thymocytes decreases by 15% after 6 h incubation suggests that the peroxynitrite decomposition products, nitrite and nitrate, play a role in damaging cells. Here we suggest that nitrite and/or nitrate, as contaminants from the synthesis of peroxynitrite or from the decomposition of peroxynitrite, are damaging species that can cause a drop in cell viability when the thymocytes are exposed to decomposed peroxynitrite. DNA single strand breaks and repair. The results of the FADU assay for peroxynitrite-induced DNA damage and subsequential repair are shown in Fig. 2. DNA single strand breaks in thymocytes exposed to 25 mM peroxynitrite for 10 min and then incubated in fresh media were detectable 5 to 10 min after the start of the incubation. During such a short interval of time 90% of the double strand DNA was damaged; the repair process started 20 min later and the DNA showed no single strand brakes 120 min later. Formation of DPXL. Isolated thymocytes were exposed for 10 min to peroxynitrite and then incubated in complete media at 377C for different intervals of time. As shown in Fig. 3, in control thymocytes the

or 360 min). The cells were then harvested by centrifugation, and the pellet was lysed with 0.2 ml of ice-cold lysis buffer, kept on ice for 10 min, and then pelleted by centrifugation (15,000g 1 25 min at 47C). The amount of DNA in the supernatant and in the pellet was determined using the diphenylamine method (32). The percentage of DNA fragmentation was calculated by dividing the amount of DNA in the supernatant by the total DNA in the sample (supernatant / pellet), multiplied by 100.

RESULTS

Cell viability. The delayed loss of membrane integrity that characterizes apoptosis was examined by the trypan blue exclusion method. The measurements were taken as described under Methods as a function of time after treatment with peroxynitrite and incubation with or without Trolox being added to the culture media. Figure 1 shows percentage viability at different incubation times. Control cells remained about 97% viable under the conditions of the incubation, and control cells posttreated with Trolox showed about the same degree of viability as controls without Trolox. Cell treatment with 25 mM peroxynitrite for 10 min caused a relatively steady decrease in viability over time, with about 65% of the cells retaining their capacity to exclude trypan blue after 6 h incubation; about 90% of the cells that were first exposed to peroxynitrite and later incubated in fresh media in the presence of 10 mM Trolox retained the capacity to exclude trypan blue after 6 h.

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FIG. 2. Plot of percentage of double-stranded DNA in rat thymocytes after treatment with 25 mM peroxynitrite versus the time of subsequent incubation in media at 377C. Thymocytes were treated with 25 mM peroxynitrite for 10 min at room temperature. Thymocytes were prepared as described under Methods, and DNA damage was measured using the FADU assay.

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FIG. 3. DNA–protein crosslinks (DPXL) were measured in rat thymocytes treated with peroxynitrite. Cells were exposed to peroxynitrite, plated in media, and then incubated in fresh media for the indicated interval of time. Trolox (10 mM) was added to the cells at the beginning of the incubation period. The cells were then collected by centrifugation, washed in PBS, and processed for the SDS-K/ assay.

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oxynitrite only. After this period the amount of malonaldehyde equivalents formed became stable, giving 0.18 mg until the sixth hour of incubation. DNA fragmentation. Figure 5 shows the time course of DNA fragmentation in cells treated with peroxynitrite or decomposed peroxynitrite for 10 min and then incubated in fresh media with or without Trolox. The DNA fragmentation caused by peroxynitrite was detectable after 1 h incubation, during which 25% of the total DNA was fragmented. The maximum amount of fragmentation, 45%, was found after 6 h incubation. The addition of Trolox to the media decreased DNA fragmentation levels down to control levels. Gel electrophoresis analysis of apoptosis. As described above, treatment of thymocytes with 25 mM peroxynitrite results in the fragmentation of DNA, characterized by the ladder pattern evidenced by agarose gel electrophoresis analysis as shown in Fig. 6A. Figure 6B shows the absence of the DNA ladder pattern when Trolox was added to the cell culture media, immediately after peroxynitrite exposure, and the cells were harvested after incubation for increasing lengths of time. Figure 7 shows the absence of the DNA ladder pattern when BHA, BHT, and DIP were added to the incubation media at 5 mM final concentration, and the cells were incubated for 6 h. DISCUSSION

background of DPXL was rather high, 9% of the total DNA content, compared to other cell types in which the background level does not exceed 2–3% (i.e., Chinese hamster ovary cells and rat nasal epithelial cells). Such a high background level of DPXL is probably a peculiarity of thymocytes. In thymocytes exposed to peroxynitrite, we found a significant 1.6-fold increase in DPXL formation after a 1-h incubation and a 2.5-fold increase after 4 h incubation. The DPXL that are observed are equivalent to 2 to 12% of the total DNA becoming bound to proteins. When Trolox was added to the media, a partial protective effect was observed. The formation of DPXL was reduced by Trolox only during the first 2 h of incubation, but the quenching effect was no longer observed after the second hour, at which time the DPXL level was comparable to that found in peroxynitrite-treated cells to which Trolox was not added. Detection of TBARS from the oxidation of membrane lipids. Figure 4 shows the formation of TBARS in control cells, in cells treated with peroxynitrite, and/or in cells incubated with Trolox. Thymocyte treatment with 25 mM peroxynitrite for 10 min caused a time-dependent increase in TBARS varying from 0.04-mg malonaldehyde equivalents during the first hour to 0.45-1g equivalents after 6 h. When Trolox was added to the media, the formation of TBARS during the first 2 h was comparable to that found in cells treated with per-

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In this study we demonstrate that a single bolus addition of 25 mM peroxynitrite induces the formation of

FIG. 4. Time course of TBARS formation in rat thymocytes after exposure to peroxynitrite (h) and the effect of Trolox (m) added to the media at the beginning of the incubation period. Unexposed cells (l) were incubated in media as control.

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FIG. 5. Time course of DNA fragmentation observed in unexposed cells (l) or cells exposed to peroxynitrite (s) or decomposed peroxynitrite (m) and then incubated in fresh media in the presence of 10 mM Trolox (n). The percentage of fragmentation was calculated using the diphenylamine assay. This method calculates fragmentation by comparing the amount of DNA in the supernatant fraction after a 15,000g centrifugation with the total DNA in the supernatant and in the pellet.

TBARS and DPXL in rat thymocytes; Trolox is able to partially quench these effects. The treatment with peroxynitrite also causes DNA single strand breaks and apoptosis, and again Trolox provides some protection. If the cells are incubated in fresh media within 2 h after peroxynitrite treatment, the DNA nicks are completely repaired. Here, we also present some preliminary results on the ability of three phenolic antioxidants, BHA, BHT, and DIP, to protect thymocytes from apoptosis mediated by peroxynitrite. DNA nicks are generally rationalized as resulting from abstraction of a hydrogen atom from the ribose moiety, opening the sugar ring and leading to strand breakage (31). During the repair process that follows peroxynitrite exposure, the nuclear enzyme poly(ADP) ribosyltransferase (PARS) is activated (15,33). This enzyme promotes DNA repair by increasing the accessibility of the damaged area to other enzymes; however, excessive activation leads to depletion of cellular energy stores (33). The ability of peroxynitrite to initiate lipid peroxidation is well documented in vivo and in vitro (9, 34–36). In our cell system, lipid peroxidation was detected by following the formation of TBARS. Malonaldehyde (MDA) and/or other aldehydes are formed in biological samples during oxidative stress and they can react

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FIG. 6. (A) Agarose gel of DNA extracted from thymocytes. Lane 1 contains the standard 1-kb DNA ladder. Lane 2 shows DNA from unexposed thymocytes (control) incubated in fresh media for 6 h at 377C. Lane 3 shows DNA from thymocytes exposed to 25 mM peroxynitrite for 10 min in phosphate buffer and immediately after incubated for 6 h at 377C in fresh media. (B) Lane 1, 1-kb DNA ladder; lane 2, DNA from control unexposed thymocytes; lanes 3–7, DNA from thymocytes exposed to peroxynitrite for 10 min and immediately after incubated at 377C in fresh media containing 10 mM Trolox for 30 min or 1, 2, 4, or 6 h.

with amino acids and DNA and introduce cross-linkages between proteins and nucleic acids. We suggest that the low-molecular-weight aldehydes that are produced during the peroxynitrite-induced oxidation of polyunsaturated lipids are responsible for the DNA– protein cross-links effect that we observe. A similar effect is seen after exposure of different cell lines to acetaldehyde (37, 38) and formaldehyde (39). However,

FIG. 7. Agarose gel of DNA extracted from thymocytes. Lane 1 contains the standard 1-kb DNA ladder; lane 2 contains DNA from unexposed cells; lane 3 contains DNA from thymocytes exposed to 25 mM peroxynitrite and immediately after incubated at 377C in fresh media; lanes 4–8 contain DNA from cells exposed to peroxynitrite and immediately after incubated for 6 h in media containing BHA (lane 4), BHT (lane 5), DIP (lane 6), Trolox (lane 7), and ethanol (lane 8). The antioxidants were added at a final concentration of 5 mM .

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SCHEME 1

the formation of DPXL can also be attributed to the depletion of cellular thiols (40). Glutathione is the predominant thiol present in many cells, and its presence seems to be a prerequisite for protection against oxidative damage as well as apoptosis (41); depletion of cellular thiols by thiol-reactive agents, for example, increases DPXL formation (40). In thymocytes exposed to 25 mM peroxynitrite for 10 min, 9% of the thiol groups are oxidized at pH 7.4 (14). The presence of DPXL, formed within the first hour after peroxynitrite treatment, may be indirectly involved in triggering apoptosis. This possibility is suggested by the fact that unrepaired DPXL may block the normal function of the nuclear matrix. Alteration of the replication and transcription processes, caused by unrepaired DPXL, can interfere with the expression of genes involved in apoptosis. The induction of apoptosis by the p53 gene in response to DNA damage, for example, has been extensively studied (20, 42, 43). In normal cells, DNA strand breaks appear to be a necessary signal for induction of a p53-mediated response (44). The possibility that unsaturated lipids in biological membranes are critical targets during apoptosis mediated by oxidative stress is supported by the results obtained here. If one of the main steps of apoptosis is progressive lipid peroxidation, then the presence of cellular or exogenously added antioxidants could prevent the apoptotic process. Indeed, we find that if the antioxidants Trolox, BHA, BHT, and DIP were added to the cell media after peroxynitrite exposure, apoptosis was inhibited. Trolox is a strong inhibitor of membrane damage (45–

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47), and it has been shown to protect cells from oxidative stress and apoptosis in vivo (48, 49). Our results demonstrate that the presence of Trolox, after exposure to peroxynitrite, reduces the formation of TBARS and DPXL, blocks DNA nicking, and prevents apoptosis in rat thymocytes. Trolox, by protecting biological membranes and calcium homeostasis, may be precluding an initiating event in apoptosis. For example, membrane damage could increase membrane permeability to small ions, which could be of critical significance in altering calcium ion homeostasis. Raised intracellular calcium levels are thought to be responsible for activation of endonucleases that fragment DNA during apoptosis (50). The evidence that Trolox, BHA, BHT, and DIP are able to prevent DNA fragmentation indicates that phenolic antioxidants, probably including vitamin E, can serve as potent inhibitors of apoptosis induced by peroxynitrite. We are currently studying the structure– activity relationship of these compounds and trying to relate their protective effect to their affinity for cell membranes during oxidative stress. A peculiar aspect of peroxynitrite-mediated cellular damage is that immediately after DNA single strand breaks are produced, DNA repair processes are induced. However, despite these repair processes, thymocytes are committed to apoptosis several hours later. This suggests that peroxynitrite can generate a lethal signal within damaged cells that is not entirely reversed by subsequent DNA repair (see Scheme 1). Phenolic antioxidants can prevent peroxynitrite-induced oxidative stress as measured by TBARS or DPLX, and these phenols also prevent

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apoptosis. This strongly suggests that oxidative stress induces apoptosis and that both oxidative stress and apoptosis are blocked in part by antioxidant defenses. ACKNOWLEDGMENT These studies were supported by a grant from the National Institute of Environmental Health Sciences of NIH, Grant ES-06754.

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