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Am J Physiol Lung Cell Mol Physiol 291: L896-L904, 2006. First published June 23, 2006; doi:10.1152/ajplung.00053.2006
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Role of substance P in hydrogen sulfide-induced pulmonary inflammation in mice

Madhav Bhatia, Liang Zhi, Huili Zhang, Siaw-Wei Ng, and Philip K. Moore

Department of Pharmacology, National University of Singapore, Singapore

Submitted 13 February 2006 ; accepted in final form 19 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have shown earlier that H2S acts as a mediator of inflammation. In this study, we have investigated the involvement of substance P and neurogenic inflammation in H2S-induced lung inflammation. Intraperitoneal administration of NaHS (1–10 mg/kg), an H2S donor, to mice caused a significant increase in circulating levels of substance P in a dose-dependent manner. H2S alone could also cause lung inflammation, as evidenced by a significant increase in lung myeloperoxidase activity and histological evidence of lung injury. The maximum effect of H2S on substance P levels and on lung inflammation was observed 1 h after NaHS administration. At this time, a significant increase in lung levels of TNF-{alpha} and IL-1beta was also observed. In substance P-deficient mice, the preprotachykinin-A knockout mice, H2S did not cause any lung inflammation. Furthermore, pretreatment of mice with CP-96345 (2.5 mg/kg ip), an antagonist of the neurokinin-1 (NK1) receptor, protected mice against lung inflammation caused by H2S. However, treatment with antagonists of NK2, NK3, and CGRP receptors did not have any effect on H2S-induced lung inflammation. Depleting neuropeptide from sensory neurons by capsaicin (50 mg/kg sc) significantly reduced the lung inflammation caused by H2S. In addition, pretreatment of mice with capsazepine (15 mg/kg sc), an antagonist of the transient receptor potential vanilloid-1, protected mice against H2S-induced lung inflammation. These results demonstrate a key role of substance P and neurogenic inflammation in H2S-induced lung injury in mice.

neurogenic inflammation; lung injury; preprotachykinin-A; transient receptor potential vanilloid

THE TOXIC EFFECTS of hydrogen sulfide (H2S) on living organisms have been recognized for nearly three-hundred years, and until recently it was believed to be a toxic environmental pollutant with minimal physiological significance. As a broad spectrum toxicant, H2S affects many organ systems, including lung, brain, kidney, etc. It is, however, now apparent that H2S is also synthesized naturally in the body from L-cysteine mainly by the activity of two enzymes, cystathionine-{gamma}-lyase (EC 4.4.1.1 [EC] ) and cystathionine-beta-synthetase (EC 4.2.1.22 [EC] ). Both enzymes are pyridoxal phosphate dependent and are expressed in a range of mammalian cells and tissues. Of these, cystathionine-{gamma}-lyase is the main H2S-synthesizing enzyme in the vasculature (2, 18, 23).

In the first report that investigated H2S as a vasodilator, it was demonstrated that H2S relaxed rat aortic tissues in vitro (11). In a subsequent study, an intravenous bolus injection of H2S was shown to transiently decrease blood pressure of rats, an effect mimicked by pinacidil [an ATP-sensitive K+ (KATP) channel opener] and antagonized by glibenclamide (a KATP channel blocker) (25). Also, the vasorelaxant effect of H2S is mainly mediated by an interaction of the gas with smooth muscle cells, and the H2S-induced vasorelaxation is dependent on the entry of extracellular Ca2+ (26).

Although the role of H2S as a vasodilator has been known for some time, the part played by H2S in inflammation has only recently begun to be addressed. In the first report on the role of endogenous H2S in inflammation, it has been shown (4) that H2S plays an important proinflammatory role of in acute pancreatitis and associated lung injury. These results also raised the possibility that H2S may exert similar activity in other forms of inflammation. In subsequent studies, we and other investigators have demonstrated an important role of H2S in carrageenan-induced hindpaw edema (5), LPS-induced endotoxemia (9, 15), and cecal ligation and puncture (CLP)-induced sepsis (24). Moreover, intraperitoneal administration of NaHS, an H2S donor, at a dose of 14 µmol/kg has been shown to induce lung inflammation by itself (15) and, at a dose of 10 mg/kg, also to worsen CLP-induced lung inflammation (24). However, no other organs showed evidence of inflammation in response to NaHS.

Substance P, a product of the preprotachykinin-A gene (PPT-A gene, also known as tachykinin-1 gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=full_report&list_uids=21333) is an important mediator of inflammation and has been shown to play an important role in inflammation in conditions such as acute pancreatitis and associated lung injury (7, 8, 13, 14) and CLP-induced sepsis (21).

Although recent studies have demonstrated that H2S plays a key role in lung inflammation of different etiologies, the mechanism by which H2S acts as an inflammatory mediator is not known. In this study, therefore, we have investigated the role of substance P in H2S-induced lung inflammation in the mouse.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal experiments. All animal experiments were approved by the Animal Ethics Committee of the National University of Singapore and carried out in accordance with the established "International Guiding Principles for Animal Research." Substance P-deficient PPT-A–/– mice were a generous gift from Prof. Allan Basbaum (University of California, San Francisco) and were bred as described previously (8). Balb/C (PPT-A+/+) mice or PPT-A–/– mice (20–25 g) were randomly assigned to control or experimental groups using 10 or more animals for each group. Animals were administered either NaHS (1, 5, and 10 mg/kg ip) or saline. Neurokinin-1 (NK1) receptor antagonist CP-96345 (2.5 mg/kg ip, Pfizer), NK2 receptor antagonist GR-159897 (0.12 mg/kg sc, Sigma), NK3 receptor antagonist SB-222200 (2 mg/kg sc, Sigma), CGRP receptor antagonist CGRP8–37 (0.06 mg/kg sc, Sigma), and transient receptor potential vanilloid-1 (TRPV1) antagonist capsazepine (15 mg/kg sc, Sigma) were administered to mice 30 min before NaHS injection. The doses of these drugs have been described in the literature and found to be effective in vivo (1, 10, 13, 17, 19). Some mice were anesthetized with the mixture of ketamine and medetomindine [0.75 ml ketamine (100 mg/ml) and 1 ml medetomindine (1 mg/ml), dissolved in 8.25 ml distilled water (7.5 ml/kg)] and were given capsaicin (50 mg/kg sc, Fluka) or vehicle 6 days before NaHS injection (16). After NaHS injection (1–6 h), animals were euthanized by an intraperitoneal injection of a lethal dose of pentobarbital sodium. Harvested heparinized blood was centrifuged and the plasma removed and stored at –80°C. Random sections of the lungs were fixed in 4% neutral phosphate-buffered formalin and then embedded in paraffin wax. Samples of lungs were snap-frozen in liquid N2 and stored at –80°C for subsequent measurement of tissue MPO activity.

Measurement of H2S concentration. Aliquots (120 µl) of plasma and lung homogenate were mixed with distilled water (100 µl), trichloroacetic acid (10% wt/vol, 120 µl), zinc acetate (1% wt/vol, 60 µl), N,N-dimethyl-p-phenylenediamine sulfate (20 µM; 40 µl) in 7.2 M HCl and FeCl3 (30 µM; 40 µl) in 1.2 M HCl. The absorbance of the resulting solution (670 nm) was measured 10 min thereafter by spectrophotometry (Tecan). H2S was calculated against a calibration curve of NaHS (3.125–100 µM). The absorbance of lung homogenate was corrected for the DNA content of tissue sample. Results of plasma H2S concentration (in µM) and lung H2S concentration (in nmol/µg) DNA were shown (15, 24).

Measurement of substance P levels. Plasma samples and lung homogenates were adsorbed on C18 cartridge columns (Bachem) as described (13, 21). The adsorbed peptides were eluted with 1.5 ml of 75% vol/vol acetonitrile. The samples were freeze-dried and reconstituted in assay buffer (13, 21). Substance P content was then determined with an ELISA kit (Bachem) according to the manufacturer's instructions and expressed for plasma (in ng/ml) and DNA (in pg/µg) for lung homogenates. Substance P can be measured in the range of 0–10 ng/ml in this assay.

Measurement of TNF-{alpha}/IL-1beta levels. Lung samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), centrifuged (2,000 g, 5 min, 4°C), and the supernatant collected. TNF-{alpha} and IL-1beta levels in the supernatant were evaluated by sandwich ELISA using DuoSet kit (R&D), according to the manufacturer's instructions. TNF-{alpha} and IL-1beta can be measured in the range of 15.6–2,000 and 7.8–1,000 pg/ml, respectively. Results were then corrected for the DNA content of the tissue sample and expressed DNA (in pg/µg).

Myeloperoxidase estimation. Neutrophil sequestration in lung was quantified by measuring tissue MPO activity (6, 7). Tissue samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), centrifuged (10,000 g, 10 min, 4°C), and the resulting pellet resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma). The suspension was subjected to four cycles of freezing and thawing and further disrupted by sonication (40 s). The sample was then centrifuged (10,000 g, 5 min, 4°C) and the supernatant used for the MPO assay. The reaction mixture consisted of the supernatant, 1.6 mM tetramethylbenzidine (Sigma), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide. This mixture was incubated at 37°C for 110 s, the reaction terminated with 2 M H2SO4, and the absorbance measured at 450 nm. This absorbance was then corrected for the DNA content of the tissue sample (12) [fold increase over control; MPO activity (in A450/µg DNA) in control samples in different experiments varied from 3.7890 ± 0.7158 to 8.8931 ± 0.3937].

Morphological examination. Paraffin-embedded lung samples were sectioned (4 µm), stained with hematoxylin/eosin, and examined with light microscopy as described (6, 7).

Drug preparation. Capsazepine was prepared with ethanol-Tween 80-saline (10:10:80, vol:vol:vol) and administered in a volume of 4 ml/kg. Capsaicin, GR-159897, SB-222200, and CGRP8–37 were dissolved in DMSO immediately before use and administered in a volume of 2 ml/kg. NaHS and CP-96345 were dissolved in saline.

Statistics. Data are expressed as means ± SE. In all figures, vertical bars denote the means ± SE, and the absence of such bars indicates that the means ± SE are too small to illustrate. The significance of changes was evaluated by ANOVA when comparing three or more groups. If ANOVA indicated a significant difference, the data were analyzed by using Tukey's method as a post hoc test for the difference between groups. A P value of <0.05 was considered to indicate a significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of administration of NaHS on plasma H2S, substance P levels, and lung MPO activity. Administration of the H2S donor NaHS caused a significant increase in plasma levels of H2S in a dose and time-dependent manner (Fig. 1, A and B). The maximum increase was found when mice were administered NaHS at a dose of 10 mg/kg and levels determined 1 h after NaHS administration. This increase coincided with the increase in plasma substance P levels (Fig. 1, C and D). When mice were administered NaHS at this dose (10 mg/kg) and euthanized 1 h thereafter, a significant increase in lung levels of H2S (50.72 ± 11.86 nmol/µg DNA; cf. 38.86 ± 8.62 nmol/µg DNA in controls; P < 0.05) and substance P (0.003967 ± 0.000386 ng/µg DNA; cf. 0.001963 ± 0.000385 ng/µg DNA in controls; P < 0.05) was also observed. Administration of the H2S donor NaHS also caused a significant increase in lung MPO activity in a dose and time-dependent manner (Fig. 1, E and F). Similar to the increase in H2S and substance P, the maximum increase in lung MPO activity was found when mice were administered NaHS at a dose of 10 mg/kg and euthanized 1 h after NaHS administration.


Figure 1
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  		Fig. 1. Effect of administration of NaHS on plasma levels of H2S and substance P and lung MPO activity. Mice (n = 10 in each group) were given an injection of NaHS [1, 5, and 10 mg/kg ip (A, C, and E) and 10 mg/kg ip (B, D, and F)]. One hour (A, C, and E) and different times (B, D, and F) after NaHS injection, mice were euthanized by intraperitoneal injection of pentobarbital sodium, and plasma levels of H2S (A and B) and substance P (C and D) and lung MPO activity (E and F) were determined as described in MATERIALS AND METHODS. Results shown are means ± SE. *P < 0.05, when NaHS-treated animals were compared with controls.

 
Effect of PPT-A gene deletion on H2S-induced lung inflammation. Evidence of lung injury induced by intraperitoneal administration of NaHS was confirmed by an increase in lung MPO as a measure of neutrophil infiltration (Fig. 2) in mice treated with NaHS compared with control mice treated with intraperitoneal normal saline. Moreover, lung levels of TNF-{alpha} and IL-1beta were also increased in mice after NaHS administration. In PPT-A–/– mice, which are genetically deficient in substance P, the effect of NaHS on lung MPO activity, TNF-{alpha}, and IL-1beta levels was significantly attenuated (Fig. 2). Histological examination of lung sections confirmed a protection by PPT-A gene deletion on lung in terms of alveolar thickening, an indicator of edema, as well as inflammatory infiltratrate (Fig. 3).


Figure 2
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  		Fig. 2. Effects of preprotachykinin-A (PPT-A) gene deletion on H2S-induced inflammation. PPT-A–/– and wild-type PPT-A+/+ mice (n = 10 in each group) were given an injection of NaHS (10 mg/kg ip). One hour after NaHS injection, mice were euthanized by intraperitoneal injection of pentobarbital sodium, and lung MPO activity (A) and TNF-{alpha} (B) and IL-1beta (C) levels were determined as described in MATERIALS AND METHODS. Results shown are means ± SE. *P < 0.05, when PPT-A–/– animals were compared with PPT-A+/+ animals.

 

Figure 3
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  		Fig. 3. Effects of PPT-A gene deletion on H2S-induced lung injury: histological evidence. A: PPT-A+/+ control. B: PPT-A–/– control. C: PPT-A+/+ mouse administered NaHS. D: PPT-A–/– mouse administered NaHS.

 
Effect of treatment with NK1 receptor antagonist CP-96345 on H2S-induced lung inflammation. To determine whether substance P was acting through NK1 receptors in H2S-induced lung inflammation, mice were treated with CP-96345, a NK1 receptor antagonist, before NaHS administration. In animals administered CP-96345, lung inflammation induced by NaHS administration was significantly reduced, as evidenced by significantly attenuated lung MPO activity, TNF-{alpha}, and IL-1beta levels (Fig. 4) and histological examination of lung sections (Fig. 5). Pretreatment with antagonists for NK2 receptors (GR-159897), NK3 receptors (SB-222200), and CGRP receptors (CGRP8–37) did not have any significant effect on H2S-induced increase in lung MPO activity (Fig. 6).


Figure 4
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  		Fig. 4. Effects of CP-96345 treatment on H2S-induced lung inflammation. Mice (n = 10 in each group) were given an injection of NaHS (10 mg/kg ip). CP-96345 was administered to mice at a dose of 2.5 mg/kg ip 30 min before NaHS injection. One hour after NaHS injection, mice were euthanized by intraperitoneal injection of pentobarbital sodium, and lung MPO activity (A) and TNF-{alpha} (B) and IL-1beta (C) levels were determined as described in MATERIALS AND METHODS. Results shown are means ± SE. *P < 0.05, when CP-96345-treated animals were compared with placebo-treated animals.

 

Figure 5
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  		Fig. 5. Effects of CP-96345 treatment on H2S-induced lung injury: histological evidence. A: control. B: mouse administered NaHS with placebo. C: mouse administered NaHS with CP-96345.

 

Figure 6
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  		Fig. 6. Effects of GR-159897, SB-222200, and CGRP8–37 treatment on H2S-induced lung MPO activity. Mice (n = 10 in each group) were given an injection of NaHS (10 mg/kg ip). Neurokinin-2 (NK2) receptor antagonist GR-159897 (0.12 mg/kg sc, Sigma), NK3 receptor antagonist SB-222200 (2 mg/kg sc, Sigma), and CGRP receptor antagonist CGRP8–37 (0.06 mg/kg sc, Sigma) were administered 30 min before NaHS injection. One hour after NaHS injection, mice were euthanized by intraperitoneal injection of pentobarbital sodium, and lung MPO activity was determined as described in MATERIALS AND METHODS. Results are means ± SE. *P < 0.05, when CP-96345-treated animals were compared with placebo-treated animals.

 
Effect of treatment with TRPV1 antagonist capsazepine on H2S-induced lung inflammation. To determine the contribution of neurogenic inflammation in H2S-induced lung inflammation, mice were treated with capsazepine, a TRPV1 antagonist, before NaHS administration. In animals administered with capsazepine, lung inflammation induced by NaHS administration was significantly reduced, as evident by significantly attenuated lung MPO activity, TNF-{alpha}, and IL-1beta levels (Fig. 7) and histological examination of lung sections (Fig. 8).


Figure 7
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  		Fig. 7. Effects of capsazepine treatment on H2S-induced inflammation. Mice (n = 10 in each group) were given an injection of NaHS (10 mg/kg ip). Capsazepine was administered to mice at a dose of 15 mg/kg sc 30 min before NaHS injection. One hour after NaHS injection, mice were euthanized by intraperitoneal injection of pentobarbital sodium, and lung MPO activity (A) and TNF-{alpha} (B) and IL-1beta (C) levels were determined as described in MATERIALS AND METHODS. Results shown are means ± SE. *P < 0.05, when capsazepine-treated animals were compared with placebo-treated animals.

 

Figure 8
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  		Fig. 8. Effects of capsazepine treatment on H2S-induced lung injury: histological evidence. A: control. B: mouse administered NaHS with placebo. C: mouse administered NaHS with capsazepine.

 
Effect of capsaicin pretreatment on H2S-induced lung inflammation. To further establish the contribution of neurogenic inflammation in H2S-induced lung inflammation, mice were treated with capsaicin before NaHS administration. In animals pretreated with capsaicin, lung inflammation induced by NaHS administration was significantly reduced, as evident by significantly attenuated lung MPO activity, TNF-{alpha}, and IL-1beta levels (Fig. 9) and histological examination of lung sections (Fig. 10).


Figure 9
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  		Fig. 9. Effects of capsaicin pretreatment on H2S-induced lung MPO activity. Mice (n = 10 in each group) were given an injection of NaHS (10 mg/kg ip). Capsaicin was administered to mice at a dose of 50mg/kg sc (or vehicle) 6 days before NaHS injection. One hour after NaHS injection, mice were euthanized by intraperitoneal injection of pentobarbital sodium, and lung MPO activity (A) and TNF-{alpha} (B) and IL-1beta (C) levels were determined as described in MATERIALS AND METHODS. Results shown are means ± SE. *P < 0.05, when capsaicin-treated animals were compared with placebo-treated animals.

 

Figure 10
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  		Fig. 10. Effects of capsaicin pretreatment on H2S-induced lung injury: histological evidence. A: control. B: mouse administered NaHS with placebo. C: mouse administered NaHS with capsaicin.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have examined the role of substance P and neurogenic inflammation in H2S-induced lung inflammation.

In a recent study, it has been shown that H2S stimulates capsaicin-sensitive primary afferent neurons in the rat urinary bladder (20), pointing to a role of H2S in neurogenic inflammation. More recently, it has been reported that relatively high concentrations of NaHS release both substance P and CGRP from guinea pig airway slices in vitro (22). In this study (22), NaHS evoked an increase in neuropeptide release in the airways that was significantly attenuated by capsaicin desensitization and by the TRPV1 antagonist capsazepine. In addition, NaHS caused an atropine-resistant contraction of isolated airways, which was completely prevented by capsaicin desensitization. We and others (4, 24) have previously shown a role of substance P and neurogenic inflammation in the pathogenesis lung inflammation of various etiologies (3), in which H2S has also been shown to play an important role. For example, the role of neurogenic inflammation in acute pancreatitis and associated lung injury was demonstrated by the use of knockout mice deficient in the gene for substance P and NKA (PPT-A gene) (8), for NK1 receptors (7), and by the use of the specific NK1 receptor antagonist CP-96345 (13, 14). Similarly, both H2S (24) and substance P (21) play a key role in the pathogenesis of lung inflammation in CLP-induced sepsis.

Administration of an H2S donor to mice resulted in an increase in plasma substance P levels in a dose and time-dependent manner. This treatment also caused lung inflammation, as evident by an increase in lung MPO activity, an increase in tissue TNF-{alpha} and IL-1beta levels, and histological evidence of lung injury. The maximum increase in plasma levels of H2S and substance P and lung MPO activity was found when mice were administered NaHS at a dose of 10 mg/kg and euthanized 1 h after NaHS administration. Therefore, this dose and time of NaHS administration were used in all subsequent experiments.

To investigate the role of substance P and NK1 receptors in H2S-induced lung inflammation, we used two different and complementary approaches: PPT-A–/– mice, which are genetically deficient in substance P, and CP-96345, an NK1 receptor antagonist. Both genetic deletion of substance P and treatment with an NK1 receptor antagonist protected mice against H2S-induced lung inflammation, as evident by a significantly attenuated increase in lung MPO activity, TNF-{alpha}, IL-1beta levels, and histological evidence of lung injury. The effect of H2S on lung inflammation (via substance P) was dependent on NK1 receptors but not NK2, NK3, or CGRP receptors, because treatment with antagonists of these receptors had no significant effect on the increase in H2S-induced lung MPO activity.

In an earlier study (22), it has been shown that H2S possesses the ability to stimulate sensory neurons and cause the release of neuropeptides also in the guinea pig airways. This conclusion was derived from three observations. First, H2S-induced release of both Substance P and CGRP-like immunoreactivities in guinea pig airways was totally prevented by capsaicin desensitization. Second, H2S-induced contraction of the isolated guinea pig bronchus or trachea was unaffected by muscarinic receptor blockade, but it was abolished or, for certain H2S concentrations, converted into relaxation by capsaicin desensitization. Third, H2S-induced contraction was abolished by blockade of tachykinin NK1 and NK2 receptors. In the current study, to investigate the contribution of neurogenic inflammation in H2S-induced lung inflammation, we again employed two alternative and complementary approaches. First, mice were chronically treated with capsaicin to ablate substance P-producing sensory nerves. In another experiment, mice were treated with capsazepine, an inhibitor of the TRPV1, the receptor for capsaicin. Both ablation of sensory nerves with capsaicin and treatment with capsazepine protected mice from H2S-induced lung inflammation, thereby showing a key role of neurogenic inflammation in H2S-induced lung injury. These observations are of particular relevance because TRPV1 undergoes remarkable sensitization/upregulation by a large variety of exogenous agents or endogenous stimuli, including activation of certain G protein-coupled receptors (bradykinin B2 receptor and proteinase-activated receptor-2) or tyrosine kinase receptor via protein kinase C or phospholipase C stimulation (22). Thus other mediators, the expression of which is increased in inflammation, may well synergize with H2S to exaggerate TRPV1 excitation on afferent and efferent discharge of sensory nerve terminals, thus aggravating the symptoms produced by sensory nerve stimulation. The precise molecular site of action of H2S, however, by which it causes neurogenic inflammation and substance P upregulation and thereby contributes to lung injury, remains to be investigated.

Nevertheless, these results show for the first time that H2S induces lung injury via neurogenic inflammation and substance P. Substance P, in turn, contributes to inflammation by acting through NK1 receptors.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Biomedical Research Council Grant R-184-000-094-305 and the Office of Life Sciences Cardiovascular Biology Program Grant R-184-000-074-712, National University of Singapore.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bhatia, Dept. of Pharmacology, National Univ. of Singapore, Yong Loo Lin School of Medicine, Bldg. MD2, 18 Medical Dr., Singapore 117597 (e-mail: mbhatia{at}nus.edu.sg)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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