Induction of apoptosis in the lung but not in the liver of rats receiving intra-tracheal instillations of chromium(VI)

Francesco D'Agostini, Alberto Izzotti, Carlo Bennicelli, Anna Camoirano, Elena Tampa and Silvio De Flora,1

Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy

Abstract

Several studies have shown that hexavalent chromium [Cr(VI)] induces apoptosis in a variety of in vitro test systems. We instilled intra-tracheally either saline or sodium dichromate (0.25 mg/kg body weight), for three consecutive days, to Sprague–Dawley rats. TUNEL analyses showed a marked increase of the apoptotic index in both bronchial epithelium and lung parenchyma of Cr(VI)-treated rats, but no effect was detected in their liver. In parallel, the expression of 13 out of 18 apoptosis-related genes, evaluated by cDNA array analysis, was significantly enhanced in rat lung. The overexpressed genes included c-Jun N-terminal kinases 1, 2 and 3, bcl-x, bcl-2-associated death promoter and bcl-2-related ovarian killer protein, caspases 1, 3 and 6, DNase I precursor, DNA topoisomerases I and II alpha, and poly(ADP-ribose) polymerase. The enhancement of p53 expression in the lung was borderline to statistical significance. Expressions of bcl-2, bax-alpha, mdm2 and DNA topoisomerase IIB were not enhanced to a significant extent in lung. No induction of gene expression was observed in rat liver. RT-PCR analyses confirmed that Cr(VI) enhances the expression of c-Jun N-terminal kinase 1, caspase 6, and DNase I precursor but not that of bcl-2 in lung, while none of these genes was overexpressed in the liver of Cr(VI)-treated rats. The lack of stimulation of apoptosis in the liver parallels the failure of Cr(VI) to produce genotoxic damage, as we previously observed under identical experimental conditions. These negative findings may be ascribed to reduction of Cr(VI) to Cr(III) when traveling from the respiratory tract to the liver. On the other hand, induction of apoptosis in the respiratory tract parallels the occurrence of genotoxic effects and oxidative DNA damage produced by Cr(VI) in the same tissue. As previously shown in another laboratory, Cr(VI) did not induce lung tumors after 30 months of administration of the same daily dose. Therefore, apoptosis is likely to provide a protective mechanism at a post-genotoxic stage of Cr(VI) carcinogenesis.

Abbreviations: Cr(VI)/Cr(V)/Cr(IV)/Cr(III), hexavalent/pentavalent/tetravalent/trivalent chromium; TUNEL, TdT-mediated dUTP nick end labeling; JNK, c-Jun N-terminal kinase; BAD, bcl-2-associated death promoter; BOK, bcl-2-related ovarian killer protein; DNase, deoxyribonuclease; mdm2, murine double minute-2; PARP, poly(ADP-ribose) polymerase; ELF, epithelial lining fluid; PAM, pulmonary alveolar macrophages; ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction.

Introduction

A quite extensive literature covers the subject of apoptosis in vitro, but less information is available in in vivo test systems. Moreover, it is surprising that far fewer papers deal with the topic of apoptosis specifically in the lung than in the other major organs, in spite of the increasing evidence that apoptosis plays a critical role in a variety of physiological and pathological processes occurring in the lung (1).

Therefore, it is of interest to evaluate the apoptotic response of the lung to pulmonary carcinogens by using suitable animal models. Previously, we detected a powerful induction of apoptosis in the lung of rats exposed to cigarette smoke (2). In the present study we investigated the effects produced by exposure of rats to hexavalent chromium [Cr(VI)], a selective lung carcinogen whose carcinogenicity is attenuated by host protective mechanisms in both humans and animals (3,4). Both soluble Cr(VI) compounds, such as potassium chromate, sodium chromate, potassium dichromate and sodium dichromate, and an insoluble Cr(VI) compound, lead chromate, were shown to induce apoptosis in cultured cells from either human or rodent sources (5–18). We administered sodium dichromate by intratracheal (i.t.) instillation, since this route bypasses proximal airways, and in particular the highly developed nasal filter of rats, thereby delivering the test agent directly to the respiratory tract. For this reason, the i.t. route has been recommended by WHO as a suitable test system for lung carcinogenesis (19).

As assessed by in situ end-labeling methodology, we detected a marked induction of apoptosis in the bronchial epithelium and lung parenchyma of Cr(VI)-treated rats, but not in their liver. In addition, as a part of a broader study evaluating multigene expression by cDNA array technology (A.Izzotti et al., manuscript in preparation), the transcription of several genes involved in the apoptotic process was significantly enhanced in the lung but not in the liver of the same animals. These patterns were confirmed when evaluating gene expression by RT-PCR.

Materials and methods

Animals and treatment
Sixteen male Sprague–Dawley rats (Harlan Italy, S. Pietro al Natisone, Italy), 6–8 weeks of age and weighing 120–140 g, were acclimatized for 10 days and divided randomly into two groups. They were maintained on a standard rodent chow (MIL, Morini, San Polo d'Enza, Italy), and given drinking water ad libitum. The animals were housed in a climatized environment with a temperature of 22 ± 1°C, relative humidity of 50 ± 5%, and ventilation accounting for 15 air renewal cycles/hour. Animal care and treatments were in accordance with our institutional and national guidelines.

All rats received daily, for three consecutive days, i.t. instillations of either 0.15 M NaCl (sham-treated rats) or sodium dichromate [Cr(VI)-treated rats]. Sodium dichromate (Na2Cr2O7.2H2O, E. Merck, Darmstadt, Germany) was dissolved in 0.15 M NaCl and administered at a dose of 0.25 mg/kg body weight/day. Administration was performed by instilling 100 µl of material via a cannula guided by a modified otoscope, which was inserted into the trachea proximally to its bifurcation into main bronchi.

The rats were deeply anesthetized with diethyl ether and killed by cervical dislocation 24 h after the last i.t. instillation. Liver and lungs were removed from each animal. The left lung and liver lobes were fixed in formalin and embedded in paraffin for assessing apoptosis, whereas the right lung and liver lobes were immediately stored at –80°C for molecular analyses.

Detection of apoptosis
Sections (5 µm) of paraffin-embedded lung and liver were cut and placed onto slides treated with poly-L-lysine (Poly-PrepTM Slides, Sigma Diagnostics, St Louis, MO, USA). Apoptotic cells were identified by TdT-mediated dUTP nick end labeling (TUNEL) method, which quantifies apoptotic cell death at the single cell level and preferentially labels apoptosis, thereby discriminating it from necrosis and from primary DNA strand breaks (20). We used the apoTACSTM-Blue Label in situ Apoptosis Detection Kit (Trevigen, Gaithesburg, MD, USA), following the manufacturer's instructions. The slides were scored at a magnification of x400, and 1000 cells/organ/rat were examined. The results are expressed as apoptotic index, indicating the % of cells affected by apoptosis.

Evaluation of multigene expression by cDNA array
Aliquots (15 µg) of liver and lung were collected and pooled within each group of either sham-treated or Cr(VI)-treated rats. RNA extraction was performed by using an automated nucleic acid extractor (Genepure 341, Applied Biosystems, Foster City, CA, USA). cDNA probes were synthesized by incubating RNA from each pool (20 µg) with reverse transcriptase (200 U, SuperScript II RT, Lifetech, Gaithersburg, MD, USA) in the presence of gene specific primers (Clontech Lab., Palo Alto, CA, USA) and AT-{alpha}-32P (Amersham, Buckinghamshire, UK). After purification on CromaSpin-200 diethyl pyrocarbamate-H2O columns (Clontech), radioactivity was measured by InstantImager (Packard, Meriden, CT, USA). The samples of liver and lung pooled from either sham-treated or Cr(VI)-treated rats were accurately equalized (18 x 106 c.p.m./sample) and mixed with Cot-1 DNA (Clontech). Salmon test DNA (Sigma) was added to cDNA expression arrays (AtlasTM Rat Stress Array, Clontech), which were then incubated overnight at 68°C with radioactive probes in a hybridization oven (Bibby Stuart, Staffordshire, UK). After six washings with SDS–SSC solutions, hybridization arrays were analyzed by InstantImager. The data were processed by AtlasTM Image software (Clontech).

The Rat Stress Array includes 216 genes, of which nine are housekeeping genes and 18 are categorized as involved in the apoptotic process. All four samples were assayed in the same experiment, in which the expression of each gene was assessed in duplicate. The ratio of signal intensity, measured for each gene, to background levels was calculated. Two separate experiments were performed. Therefore, the results are means ± SD of four data.

Evaluation of gene expression by RT-PCR
The expression of JNK1, bcl-2, caspase 6, and DNase I precursor genes was further evaluated by using semiquantitative mRNA reverse transcription (RT) followed by PCR cDNA amplification. Gene specific mRNA antisense primers, available from GenBank database (www.ncbi.nlm.nih.gov) were used to synthesize cDNAs by incubating 5 µg RNA with 100 U reverse transcriptase (Invitrogen, Carfsbad, CA, USA) at 42°C for 50 min. cDNA was amplified by Taq polymerase (Invitrogen) reactions. The number of PCR cycles was optimized for each gene in order to ensure that product intensity fell within the linear phase of amplification. Specific reaction conditions, as related to primer sequences, were set up according to a commercially available software (Primer Premier 4, Premier Biosoft International, Palo Alto, CA, USA). Reaction products were separated by agarose gel electrophoresis and identified by comparison with an 800–50 bp DNA ladder (Invitrogen). cDNA amounts were quantified by densitometric analysis using a digital acquisition equipment (DC 120 Zoom Digital Camera, Eastman Kodak, Rochester, NY, USA) and a specifically designed software (1D Image Analysis Software, Eastman Kodak). The results were expressed in terms of arbitrary units, which indicate the intensity of fluorescence emitted by DNA-bound ethidium bromide after subtracting background levels. Three separate experiments were run for each gene.

Statistical analysis
The significance of differences between sham-treated and Cr(VI)-treated rats, regarding the apoptotic index (means ± SD of eight rats) and the expression of each gene (means ± SD of four data for cDNA arrays and three data for RT-PCR analyses), was evaluated by Student's t test for unpaired data.

Results

The photographs shown in Figure 1Go provide examples of the results obtained by TUNEL method. The mean (±SD) values of apoptotic index recorded among the eight rats belonging to each experimental group are also reported. There was no difference between sham-treated and Cr-treated rats in liver cells, while the apoptotic index grew 6.7-fold (P < 0.001) in the bronchial epithelium and 22.1-fold (P < 0.0001) in the lung parenchyma following exposure to Cr(VI).



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Fig. 1. Selective induction of apoptosis, as evaluated by TUNEL method, in the bronchial epithelium, lung parenchyma, and liver cells of Sprague–Dawley rats receiving i.t. instillations either of saline (Sham) or of sodium dichromate [Cr(VI)] (original magnification x200). The apoptotic index (mean ± SD of eight rats) recorded in each treatment group and tissue is also shown.

 
Table IGo reports the results of cDNA array analyses evaluating the expression of 18 genes involved in the apoptotic process. The genes are ordered according to the AtlasTM gene code coordinates. The GenBank accession codes and the coded proteins are also indicated. The expression of all genes was very similar in the liver of sham-treated and Cr(VI)-treated rats, with no significant difference between the two groups, and with a Cr(VI)/Sham ratio ranging between 0.7 and 1.3. In contrast, the expression of 13 genes was significantly increased in the lung following exposure of rats to Cr(VI), with Cr(VI)/Sham ratios up to 2.6. The overexpressed genes encoded for JNK1, JNK2 and JNK3; bcl-x, BAD and BOK; caspases 1, 3 and 6; DNase I precursor; DNA topoisomerases I and II alpha; and PARP. Gene expression was not enhanced to a significant extent for bcl-2 and bax-alpha; p53 nuclear oncoprotein and mdm2 p53-associated protein; and DNA topoisomerase IIB.


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Table I. Expression of 18 genes involved in the apoptotic process in liver and lung of Sprague–Dawley rats receiving intra-tracheal instillations either of saline (Sham) or of Cr(VI), as assessed by cDNA array technology
 
The expression of four of the above mentioned genes was also evaluated by RT-PCR. The optimal numbers of PCR cycles needed to obtain linear amplifications were 48 for JNK1 (389 base pairs), 65 for bcl-2 (969 base pairs), 54 for caspase 6 (781 base pairs), and 48 for DNase I precursor (90 base pairs). Figure 2Go provides examples of the results obtained by RT-PCR analysis of the expression of these genes. Table IIGo reports the means (±SD) of the results obtained in three separate experiments. Also by using RT-PCR, the expression of all four tested genes was very similar in the liver of sham-treated and Cr(VI)-treated rats, with no significant difference between the two groups, and with a Cr(VI)/Sham ratio between 1.0 and 1.2. The expression of JNK1, caspase 6, and DNase I precursor was significantly increased in the lung following exposure of rats to Cr(VI), with Cr(VI)/Sham ratios of 1.5, 1.4, and 1.5, respectively. In agreement with the results of cDNA array analyses, evaluation by RT-PCR of bcl-2 expression did not show significant increases in the lung.



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Fig. 2. Analysis by RT-PCR of the expression of four genes in lung and liver of Sprague–Dawley rats receiving i.t. instillations either of saline (Sham) or of sodium dichromate [Cr(VI)]. The lane on the left shows a DNA ladder.

 

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Table II. Expression of four genes involved in the apoptotic process in liver and lung of Sprague–Dawley rats receiving intra-tracheal instillations either of saline (Sham) or of Cr(VI), as assessed by RT-PCR
 
Discussion

The results of the present study provide evidence that a short-term exposure of rats, by the i.t. route, to rather high doses of Cr(VI), which would correspond to 17.5 mg/day for a 70 kg man, induces the apoptotic process in the respiratory tract but not in liver cells.

The lack of induction of apoptosis in the liver, as assessed by TUNEL method, cDNA array technology, and RT-PCR, parallels the findings that Cr(VI) fails to produce DNA damage (21) and to induce the transcription of a large number of genes involved in cellular functions other than apoptosis (A.Izzotti et al., manuscript in preparation). Since 51Cr is distributed to the liver after i.t. administration (22), these patterns support the view that Cr(VI) is detoxified when traveling at a distance from the portal of entry into the organism. In fact, Cr(VI) carcinogenicity to humans is restricted to the respiratory tract (4,23–25), and tumorigenicity to experimental animals was just detected in a minority of the studies available in the literature, and only occurred at implantation sites (4,23). From a mechanistic point of view, efficient detoxification of Cr(VI) following airborne exposure occurs as a consequence of reduction to inactive trivalent chromium [Cr(III)] first in the respiratory tract, e.g. in the epithelial lining fluid (ELF), pulmonary alveolar macrophages (PAM), bronchial cells, and lung parenchyma cells, then in circulating blood, where Cr(VI) is rapidly uptaken by erythrocytes, therein reduced to Cr(III) and bound, mainly to hemoglobin, and finally in the liver itself (3,4). These mechanisms have been extensively investigated in our laboratory for nearly 25 years in ex vivo studies using body fluids, cells, tissues, and organs from several animal species, also including humans (3,4,26). Quantitative estimates of Cr(VI) reduction in various body compartments have been provided (3,4). It is meaningful that apoptosis in the liver periportal region had only been detected in BDF1 mice receiving injections of huge amounts of sodium dichromate, i.e. 25 mg/kg body weight (27). This would correspond to the extraordinarily high amount of 1.75 g Cr(VI) for a 70 kg man. Clearly, these experimental conditions do not mimic any human exposure, by-pass toxicokinetic processes, and overwhelm the metabolic defense capability of the host organism.

In contrast, we found that the i.t. instillations of Cr(VI) stimulated apoptosis in the rat respiratory tract. First of all, there was a significant and considerable increase of the apoptotic index, as assessed by TUNEL method, in the bronchial epithelium and, even more sharply, in the mixed-cell population of lung parenchyma. In addition, the results of cDNA array and RT-PCR analyses were consistent with overexpression of several genes which are known to play a role in the apoptotic cell death. Clearly, due to the extreme complexity of the apoptotic process, even the simultaneous evaluation of 18 genes in a single study offers a partial and fragmentary view of the network of events which ultimately lead to programmed cell death. Nonetheless, the data provided by these molecular analyses are useful to support the results of in situ end-labeling methodology and elucidate some mechanisms which are specifically triggered by Cr(VI) in vivo.

Although p53-independent pathways do exist, this gene is well known to play a key role in the apoptotic signaling consequent to genotoxic damage. The expression of this gene was not significantly enhanced in the lung of Cr(VI)-treated rats. It is noteworthy that in vitro studies had indicated that activation of p53 in human lung epithelial A549 cells occurs at the protein level rather than at the transcriptional level (28). It was suggested that p53 is involved in late stages of Cr(VI)-induced apoptosis in these cells (12). However, it should be noted that these studies (12,28) employed extremely high doses of Cr(VI) and malignant lung tumor cells. The p53-dependent nature of Cr(VI)-induced apoptosis is also supported by the evidence that the p53 oncoprotein was translocated from the cytoplasm to the nucleus after treatment of diploid human lung fibroblasts (HLF cells) with Cr(VI) (16). Furthermore, the oral administration of Cr(VI) to p53-deficient C57BL/6TSG-p53 mice resulted in an increased toxicity to the liver, compared with isogenic, p53-proficient mice (29). In parallel, our data showed a modest and not significant increase of mdm2 gene expression in the lung of Cr(VI)-treated rats. This gene provides a negative autoregulatory feed-back mechanism by binding to p53 and blocking its transcriptional activity. Moreover, mdm2 shuttles the p53 oncoprotein out of the nucleus and mediates p53 degradation by ubiquitin-protease (28). Treatment of A549 cells with Cr(VI) decreased the in vitro binding of mdm2 to p53 (28).

The expression of all three monitored JNKs was significantly enhanced in rat lung following exposure to Cr(VI). Although the role of JNKs in death receptor-mediated apoptosis is still unclear, these kinases have been shown to be induced by a variety of apoptotic stimuli in different cell types (30). Activation of the JNK pathway by Cr(VI) also occurs in human non-small cell lung carcinoma CL3 cells (31).

Within the bcl-2 family, treatment of rats with Cr(VI) significantly enhanced expression in the lung of the two pro-apoptotic genes BAD and BOK. The expression of the anti-apoptotic gene bcl-2 was not enhanced to a significant extent, whereas a marked overexpression was observed with bcl-x. The latter gene behaves like a double-edged sword, since it can be transcribed into two mRNAs through alternative splicing. The resulting protein products are bcl-xl and bcl-xs, which function as apoptosis repressor and accelerator, respectively (32).

The expression of all three monitored caspases was significantly enhanced by Cr(VI) in rat lung. These cysteine-dependent aspartate-specific proteases belong to the sub-category of effector caspases, which are responsible for dismantling cellular proteins (33). Substrates for caspases, including PARP and two of the three analyzed DNA topoisomerases (I and II alpha), were also overexpressed. A marked increase occurred with DNase I precursor. Caspase-activated DNases, such as DNase I, degrade nuclear DNA into nucleosomal units, which is one of the hallmarks of apoptotic cell death (34).

After entering the cells through the general anion channel protein (band 3 protein), as chromate oxyanion, Cr(VI) tends to be reduced in different cell compartments via a network of mechanisms, both non-enzymatic and enzyme-catalyzed. These processes lead to generation of Cr(V), Cr(IV), Cr(III), and reactive oxygen species (ROS). ROS and Cr(III), the stable reduced species, are likely to ultimately cause genotoxic effects in Cr(VI)-exposed cells (reviewed in ref. 35). It has also been suggested that Cr(VI) reduction directly oxidizes DNA phosphate and bases (36). Under experimental conditions which were identical to those designed for the present study, we previously found that Cr(VI) causes DNA fragmentation, DNA–protein crosslinks, and 32P post-labeled nucleotide modifications in lung cells (21). In addition, there was an increase in 8-hydroxy-2,3-deoxyguanosine (8-OH-dG) in lung DNA, which documented the in vivo induction of oxidative DNA damage following treatment with Cr(VI) (21). Interestingly, ROS have been shown to be associated with induction of apoptosis by Cr(VI) in cultured mammalian cells (10,12,15).

As shown in another laboratory (37), the i.t. instillation of sodium dichromate to Sprague–Dawley rats, at the same dose (0.25 mg/kg body weight), 5 times a week for 30 months, did not induce lung tumors. Sodium dichromate elicited a weak carcinogenic response only when the same cumulative amount of compound (1.25 mg/kg body weight) was given once weekly in a single dose (37), which suggests that a single massive dose could at least partially overwhelm the defense mechanisms of the respiratory tract. Our studies provided evidence that toxicokinetic and metabolic factors imprint a threshold character to Cr(VI) carcinogenesis (reviewed in refs 3 and 4), a view which was shared by agencies, such as IARC (23) and the US Department of Health and Human Services (25).

Focusing on the respiratory tract, protective barriers are provided by the powerful aspecific defense mechanisms of airways and, specifically, by Cr(VI) reduction in extracellular fluids (ELF) and long-lived sweeping cells (PAM) (38). Moreover, even within target cells, reductive metabolism can lead to detoxification. In fact, after Cr(VI) uptake, generation of reactive derivatives in proximity of DNA represents an activation process, whereas reduction in other cell compartments results in trapping of reactive derivatives by a myriad of cell ligands, thereby providing a detoxification process (10,35). As a further support to this interpretation, the i.t. administration of the same dose of Cr(VI), five times a week for 4 weeks, resulted in an enhanced ability of the lung to metabolize Cr(VI) itself (39), a finding which correlates with its lack of carcinogenicity (37).

In the rat model employed in this study, however, toxicokinetic and metabolic mechanisms were not sufficient to explain the discrepancy between the short-term induction of genotoxic damage in lung cells (21) and the failure of Cr(VI) to induce lung tumors in the long term, under the same experimental conditions (37). Therefore, the clue for this discrepancy should be sought downstream of the genotoxic and oxidative damage. Apoptosis is believed to provide an important mechanism favoring the death and removal of DNA damaged cells, which prevents further evolution towards cancer. This conclusion was drawn by using in vitro test systems (10) and is further supported by our in vivo data, which show the selective induction of apoptosis by Cr(VI) in rat lung, accompanied by modulation of the expression of several genes involved in apoptotic cell death. In addition, it is noteworthy that cDNA array analyses showed that, besides overexpression of apoptosis-related genes, the i.t. instillations of Cr(VI) enhanced at least twice the expression of other 92 genes in rat lung (A. Izzotti et al., manuscript in preparation). These included not only genes specifically involved in the metabolic reduction of Cr(VI) but also genes encoding for other cellular functions which are strictly interconnected with apoptosis, such as response to stress, protein and DNA repair mechanisms, signal transduction pathways and regulation of cell cycle.

In conclusion, the findings of the present study show that Cr(VI) can induce apoptosis and regulate the expression of genes involved in this process in the rat respiratory tract, where this metal species induces oxidative damage and genotoxicity but fails to exert tumorigenic effects under the same experimental conditions.

Notes

1 To whom correspondence should be addressed
Email:sdf{at}unige.it Back

Acknowledgments

This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).

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Received August 8, 2001; revised December 6, 2001; accepted December 14, 2001.