ELSEVIER European Journal of Pharmacology

Environmental Toxicology and Pharmacology Section 292 (1994) 75-80

ejp environmental toxicology

and pharmacology

In vivo administration of taurine and niacin modulate cyclophosphamide-induced lung injury

N a r a y a n a n V e n k a t e s a n , G o w r i C h a n d r a k a s a n *

Department of Biochemistry, Central Leather Research Institute, Adyar, Madras-600 020, India

Received 3 March 1994; revised MS received 15 April 1994; accepted 19 April 1994

Abstract

The antiinflammatory, antioxidant activity of taurine and niacin against cyclophosphamide-induced early lung injury in rats was investigated. A single intraperitoneal injection of cyclophosphamide markedly altered the levels of several biomarkers in bronchoalveolar lavage fluid: total protein, albumin, angiotensin converting enzyme, lactate dehydrogenase, lactate, N-acetyl-/~- o-glucosaminidase, alkaline phosphatase, acid phosphatase and lipid peroxidation product were significantly elevated. In contrast, decreased levels of total reduced glutathione (GSH) and ascorbic acid were observed. Cyclophosphamide significantly increased malondiaidehyde levels in serum and lung. Significant increases in lung content of lipid hydroperoxides were seen that paralleled the decreased levels of total reduced glutathione and total sulfhydryl groups. Pretreatment of rats with daily intraperitoneai injection of taurine plus niacin 7 days prior to and 2 days after cyclophosphamide insult significantly inhibited the development of lung injury, prevented the alterations in lavage fluid biomarkers associated with inflammatory reactions, with less lipid peroxidation and restoration of antioxidants. In conclusion, our results suggest that taurine and niacin in combination is efficient in blunting cyclophosphamide-induced pulmonary damage.

Keywords: Cyclophosphamide; Taurine; Niacin; Lung injury antioxidant; Membrane stabilizer

I . I n t r o d u c t i o n .

Chemotherapy with a cytotoxic and immunosuppres- sive drug like cyclophosphamide has been associated with the development of lung fibrosis both in animals and humans (Gould and Miller, 1975; Collis et al., 1980; Batist and Andrews, 1981; Cooper et al., 1986; Kanekal 'et al., 1992). Histopathological lesions charac- teristic of cyclophosphamide toxicity include endothe- lial cell damage, epithelial cell abnormalities with desquamation of type I pneumocytes, intraalveolar ex- udation, interstitial inflammation, fibroblast prolifera- tion with fibrosis (Gould and Miller, 1975). The mecha- nism(s) which result in cyclophosphamide-induced lung injury are, however, unclear and there is increasing evidence that reactive oxygen species can be of impor- tance in the pathophysiology of pulmonary injury asso- ciated with the inflammatory responses (Patel, 1990).

*Corresponding author. Tel.: (91)-0aA 412616; Fax: (91)-044- 411589.

0926-6917/94/$07.00 O 1994 Elsevier Science B.V. All rights reserved SSDI 0926-6917(94)00022-8

Much of the ongoing research is devoted to the poten- tial use of new pharmacological compounds that pre- vents the deleterious effect of these toxic oxygen species. Moreover, it was shown in experimental mod- els that several antiinflammatory, antioxidant com- pounds, inspite of their diverse structures, were all capable of reducing the severity of drug-induced pul- monary injury (Thrall et al., 1979; Pepin and Langer, 1985; Hoffer et al., 1993). Recent studies suggest that taurine, 2-aminoethane sulfonic acid, an abundant free amino acid in the cytosol of all organs, as an antiin- flammatory, antioxidant, membrane stabilizer (Naka- shima et al., 1983; Pasantes-Morales et al., 1985; Wright, 1986; Gordon et al., 1986) and niacin, a water- soluble vitamin B, as a precursor of nicotinamide ade- nine dinucleotide involved in the DNA repair (Bender et al., 1982; Weitberg, 1989) have a protective role in ameliorating pulmonary injury in animal models, par- tieularly following treatment with bleomyein (Wang et al., 1989, 1990, 1991, 1992) and amiodarone (Wang et al., 1992). A previous study from our laboratory (Ven- katesan and Chandrakasan, 1993) also shows that s u c h

76 N. 1/enkatesan, G. Chandrakasan / Eur..L PharmacoL Enriron. Toxicol. Phannacol. Section 292 (1994) 75-80

a treatment significantly inhibits the development of early lung injury following paraquat administration to rats. It was the intention of this investigation to provide additional support that taurine and niacin treatment reduced the development of early lung injury in cyclo- phosphamide-exposed rats. It is well established that in experimental models of pulmonary fibrosis, the se- quence of physiologic and histoiogic changes consists of an acute inflammatory response and altered levels of lavage fluid biomarkers concurrent with acute pul- monary injury (Hampson et al., 1989; Schultze et al., 1991); changes in these biomarkers also occur following cyclophosphamide intoxication (Venkatesan and Gowri Chandrakasan, 1994). This study was therefore initi- ated to determine whether taurine and niacin could alter cyclophosphamide-induced changes in lavage fluid biochemical constituents including oxidative damage.

under light ether anaesthesia and lung lavage was performed. Approximately 5 ml of blood was collected, allowed to clot and the serum separated. The lungs were then dissected free, chilled in ice cold physio- logical saline (0.9% w/v) and weighed.

2.3. Collection of bronchoah,eolar lat'age fluid

Lung lavage was performed 24 h after the experi- mental period. Six rats in each experimental groups were killed and the thoracic viscera was exposed. The trachea was cannulated and the lung was infused 5 times with ice cold, sterile, physiological saline at a volume of 3 ml per wash. The lung lavage fluid from each rat was centrifuged at 300×g for 10 min to sediment the cells prior to biochemical analyses.

2. 4. Biochemical assays

2. M a t e r i a l s a n d m e t h o d s

2.1. Materials

Cyclophosphamide (Endoxan Asta) was purchased from Khandelwal Laboratories, India. Bovine serum albumin, taurine, niacin, lithium lactate, Triton X-100, phenylphosphate disodium salt, p-nitrophenyl-N- acetyl-fl-o-glucosaminide, and thiobarbituric acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Hippuryl-L-histidyl-L-leucine was synthesized and purified in Chemical Laboratory, Central Leather Research Institute, Madras, India. All other chemicals w e r e of analytical grade and commercially available.

2.2. Experimental design

Male Wistar rats weighing 100-110 g were used in the present experiment. Rats were housed and main- tained in polyvinyl cages and allowed free access to food and water. The rats were divided into four experi- mental groups, each consisting of six animals (Table 1). The a n i m a l s w e r e observed daily and 24 h after the experimental period, rats were killed by decapitation

Protein content of lavage fluid and lung tissue was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Albumin con- tent was estimated by the method of Varley (1988). Angiotensin converting enzyme activity was measured using a synthetic substrate, hippuryi-L-histidyl-L-leu- cine, according to the method described by Cushman and Cheung (1971). Lactate dehydrogenase activity was determined spectrophotometrically as described by King (1965). Lactate content of lavage fluid super- natant was determined via the method of Baker and Summerson (1941). N-acetyl-/3-D-glucosaminidase ac- tivity was estimated utilizing the method described by Moore and Morris (1982). Alkaline phosphatase and acid phosphatase activities were assayed by the method of King (1965) using disodium phenyl phosphate as the substrate. Aliquots of serum and lavage fluid super- natant were incubated with two different concentra- tions of hydrogen peroxide. Malondialdehyde, a prod- uct of polyunsaturated fatty acid peroxidation was then measured as thiobarbituric acid derivative in the super- natant following precipitation with trichloroacetic acid (Cynamon et al., 1985). Total reduced glutathione con- tent of lavage fluid and lung tissue was measured based

Table 1 Treatment schedule and dosage regimen

Group Treatment with

Sterile water Cyclophosphamide Taurine Niacin (20 mg/100 g) (50 rag/100 g) (50 rag/100 g)

I (Sterile water) + - - - II (Sterile water + taurine + niacin) a + _ + + III (Cyclophosphamide) b _ + _ _ IV (Cyclophosphamide + taurine + niacin) c _ + b + +

e Treatment with water plus taurine plus niacin for 9 days; treatment with cyclophosphamide as a single dose; c pretreatment with taurine plus niacin for 7 days prior to cyclophosphamide and 2 days after cyclophosphamide. Biochemical analyses were performed on day I0 as described in Materials and methods, n ffi 6 animals in each group.

N. Venkatesan, G. Chandrakasan / Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 292 (1994) 75-80 77

Table 2 Effect of taurine a n d / o r niacin treatment on total protein, albumin, angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), lactate and N-acetyl-fl-D-glucosaminidase (NAG) levels in bronchoalveolar lavage fluid of cyclophosphamide administered rats

Treatment groups Total protein Albumin ACE LDH Lactate NAG (/~mol/100 ( tzg/ml) ( tzg/ml) (Uni t s /ml) ( IU/ l i t ) ( # m o l / m l ) mg protein)

I 178 :t: 8.53 60 -1- 5.34 14.85 5:3.14 43.64 + 4.50 1.79 + 0.13 3.51 + 0.73 II 184 + 11.31 63 -t- 6.56 15.06 + 2.08 46.18 + 3.07 1.75 + 0.013 3.60 + 0.45 III 290 + 12.85 " 104 5:7.65 " 43.45 5:5.84 " 61.36 + 6.68 a 2.60 5:0.13 a 7.35 -1- 2.06 " IV 200 + 5.14 b 72 + 6.05 b 22.63 5:2.98 c 44.85 + 3.07 b 1.95 5:0.09 b 4.85 -1- 1.58 ¢

Values are given as means 5: S.D. of six observations and the significance was tested by one way analysis of variance. a Significantly higher (P < 0.001) than all groups; b significantly lower (P < 0.01) than group III, but not significant when compared to groups I and II; c significantly lower (P < 0.01) group II1, but higher (P < 0.01) than groups I and II.

on the reaction of reduced glutathione with 5,5'-di- thiobis-(2-nitrobenzoic acid) to give a compound that absorbs at 412 nm, as with reduced glutathione which was used as a standard. Although glutathione was measured based on the DTNB method, this method is not specific and detects all sulfhydryl groups. Reduced glutathione can also be estimated enzymatically using glutathione-S-transferase. However, it is difficult to determine when the enzymatic reaction has reached completion. Therefore, in the present study we have utilized the DTNB assay for measuring glutathione, which is more rapid and sensitive (Moron et al., 1979). Ascorbic acid content of lavage fluid was analyzed by the method of Omaye et al. (1979). Lung tissue lipid peroxidation was assayed as malondialdehyde equiva- lents (Hogberg et al., 1974). Lipid hydroperoxide con- tent of lung tissue was determined by the method of Buege and Aust (1978). Lung tissue total sulfhydryl content was determined following the procedure adopted by Sed[ak and Lindsay (1968).

2.5. Statistical analysis

One way analysis of variance (ANOVA) was utilized to determine the significance of differences (P < 0.01, P < 0.00l, P < 0.0001) among group means.

3. Results

Table 2 indicates the changes in lavage fluid bio- chemical constituents which occured following cyclo- phosphamide administration. Increased concentration of total protein, albumin, angiotensin converting en- zyme, lactate dehydrogenase, lactate and N-acetyl-/i- D-glucosaminidase were evident in lavage fluid of cyclophosphamide group that received no drug treat- ment. It was of interest to note that combined treat- ment of taurine and niacin to cyclophosphamide rats almost completely prevented the increase in lavage fluid biomarkers, although these values were slightly higher than those in control groups.

Cyclophosphamide-administered rats that received no drug treatment had a significant increase in the activities of alkaline phosphatase and acid phos- phatase, increased lipid peroxidation levels, decreased levels of total reduced glutathione (GSH) and ascorbic acid (Table 3). However, these changes were remark- ably blocked by combined treatment of taurine and niacin.

The concentration of lipid peroxidation products in serum and lung (Table 4) of eyclophosphamide groups was significantly elevated from control values. The levels of lipid hydroperoxides in the lung tissue of

Table 3 Effect of taurine a n d / o r niacin treatment on alkaline phosphatase (AKP), acid phosphatase (ACT) activities, lipid peroxidation product (LPO), reduced glutathione (GSH) and ascorbic acid concentrations in lung lavage fluid of cyelophosphamide administered rats

Treatment groups AKP ACP LPO (nmol GSH Ascorbic ( IU/ l i t ) ( IU/ l i t ) malondialdehyde/ml) (p .mol /ml) acid (p .g /ml)

I 1.32 + 0.12 2.25 + 0.15 0.53 + 0.05 0.39 + 0.03 12.13 + 3.14 II 1.36 + 0.17 2.20 + 0.09 0.50 + 0.04 0A2 + 0.04 13.00 + 2.45 III 2.79 + 0.36 s 3.70 + 0.29 a 0.85 + 0.06 s 0.14 -1- 0.02 b 7.10 + 1.05 b IV 1.74 + 0.25 c 2.60 + 0.20 c 0.62 + 0.05 c 0.32 + 0.04 d 11.38 + 3.56 d,NS

Values are given as means + S.D. of six experiments. Significance between the groups was tested by one way analysis of variance, a Sign~cantly higher ( P < 0.0001) than all groups; b significantly lower ( P < 0.0001) than all groups; c significantly lower ( P < 0~001) than group HI, but higher ( P < 0.01) than groups I d and II; significantly higher ( P < 0.001) than group HI; NS not significant when compared to groups I and IT.

78 N. Venkatesan, G. Chandrakasan / Fur. J. Pharmacol. Ent,iron. Toxicol. Pharmacol. Section 292 (1994) 75-80

Table 4 Effect of taurine a n d / o r niacin treatment on serum malondialdehyde (MDA) levels, and malondialdehyde, lipid hydroperoxides, reduced glutathione and total sulfhydryl content in lungs following cyclophosphamide administration

Treatment groups Serum lipid peroxide Lung lipid peroxide Lipid Glutathione (nmol (nmol/malondialdehyde hydroperoxides (nmol /g wet issue) malondialdehyde/ml) per mg protein) /.tg of t-butyl

hydroperoxide/mg protein)

Total sulfhydryl (p.g S H / m g protein)

I 2.34 + 0.59 1.29 5:0.14 0.93 5:0.05 1.69 5:0.59 4.36 5:1.07 II 2.25 5:0.36 1.30 5:0.30 0.85 5:0.07 1.70 5:0.35 4.45 5:0.98 III 6.75 5:0.96 " 4.38 5:0.45 " 3.02 5:0.25 " 0.63 + 0.29 b 1.94 5:0.53 b IV 3.45 5:0.70 c 2.05 5:0.30 c 1.34 5:0.18 c t.16 5:0.50 d 3.68 5:0.89 d

Values are given as means-I-S.D, of six determinations. Significance between the groups was tested by one way analysis of variance. " Significantly higher (P < 0.0001) than all groups; b significantly lower (P < 0.0001) than all groups; c significantly lower (P < 0.001) than group III, but higher ( P < 0.001) than groups 1 and II; d significantly higher (P < 0.001) than group III, but lower (P < 0.01) than groups i and II.

cyclophosphamide rats were also elevated from the values obtained in the control groups. On the contrary, decreased levels of reduced glutathione and total sulfhydryl groups were observed in lungs of cyclophos- phamide-exposed rats (Table 4). These alterations at- tributable to cyclophosphamide were found to be re- duced in animals receiving taurine plus niacin.

4. Discussion

An initial objective of the present investigation was to evaluate and assess the role of taurine and niacin in modulating the onset of cyclophosphamide-induced early lung damage by analyzing bronchoalveolar lavage fluid biomarkers. A further objective of this study was to examine the ability of these compounds to decrease the oxidant burden following cyclophosphamide expo- sure. In the present study, the administration of a single intraperitoneal dose of cyclophosphamide (20 mg/100 g body weight) to rats resulted in significant pulmonary changes. Although the present dose used seems to be higher than the usual human dose, pa- tients who developed interstitial pneumonitis and pul- monary fibrosis while on cyclophosphamide therapy for chronic lymphocytic leukemia (Patel et al., 1976) and lymphoma (Spector et al., 1979) at multiple higher doses are well documented. Since the pulmonary mor- phologic and biochemical alterations induced by cyclo- phosphamide were similar to those seen in man, it is possible to extrapolate the in vivo findings from animal studies reported here to reactions in humans, provided that the mechanisms by which pulmonary damage is induced is clearly understood.

The results which we observed following cyclophos- phamide administration to rats reveal alterations in several markers of lung injury in lung lavage fluid. The key feature of this investigation was the ability of taurine and niacin in combination, to serve as an inhibitor of eyelophosphamide-indueed lung injury. It is

well known that accumulation of inflammatory leuko- cytes and activated macrophages in the alveolar region of the lung, is the initiating event during an acute inflammatory response and these cells secrete pro- teases and reactive oxygen intermediates at sites of inflammation which are implicated in the degradation of lung connective tissue components (Fox et al., 1981; Gadek et al., 1984; Weiss, 1989). Cyclophosphamide exposure could have contributed to such a mechanism, producing an acute damaging phase in the lung, char- acterized by alterations in the microvasculature brought about by the release of inflammatory mediators with increased alveolar capillary membrane permeability (Cooper et al., 1986). The principal mechanism by which taurine and niacin contribute to the blunting effect on cyclophosphamide-induced inflammatory re- actions is through their ability to reduce the migration of neutrophils and macrophages in the inflamed re- gion, thereby mitigating tissue damage and epithelial cell necrosis caused by the release of reactive oxygen species, lysosomal proteases, cytokines and other in- flammatory mediators. These results strengthen previ- ous findings that taurine and niacin treatment sup- pressed bleomycin-, amiodarone- and paraquat-in- duced lung injury. The data reported here reinforce the idea that the basis for the lesser elevation of the lavage fluid biomarkers in taurine plus niacin-treated cyclophosphamide rats may be associated with the an- tioxidant, membrane-stabilizing property of taurine and the capacity of niacin to maintain the epithelial struc- tural integrity (Wang et al., 1989, 1990, 1991, 1992; Venkatesan and Chandrakasan, 1993).

Oxidative stress associated with cyclophosphamide exposure caused a marked increase in malondialde- hyde levels in serum, lavage fluid and lung tissue. We also found cyclophosphamide administration resulted in increased concentrations of lipid hydroperoxides in lung, whereas antioxidants such as total reduced glu- tathione (GSH), total sulfhydryl groups and ascorbic acid were reduced. Available evidence suggests that

N. l/enkatesan, G. Chandrakasan / Eur. J. Pharmacol. Era'iron. Toxicol. Pharmacol. Section 292 (1994) 7.7-80 79

oxygen-free radicals are known to damage biologic systems in vivo (Halliwell et al., 1992) and these reac- tions may also be exacerbated by the reduced levels of antioxidants (Cantin et al., 1989) as observed in the present study. Pretreatment with taurine plus niacin blocked the oxidative damage associated with cyclo- phosphamide intoxication. These compounds inhibited the elevated levels of malondialdehyde and hydro- peroxides in the lung tissue. Although we used two chemically dissimilar compounds for reducing oxidative damage, the ability to inhibit lipid peroxidation and protect membranes from the damaging effect of oxygen radicals is exerted solely by taurine and there is no similar explanation for the antioxidant property of niacin. This implication was suggested by recent works on the protective effect of taurine against oxidative damage in many tissues including the lung, as well as by in vitro studies (Jacobsen and Smith, 1968; Nakashi- ma et al., 1983; Pasantes-Morales et al., 1985; Wright, 1986; Gordon et al., 1986; Huxtable, 1987; Hamaguchi et al., 1988; Wang et al., 1989; Trachtman et at., 1993). It has also been demonstrated (Banks et al., 1989) that alveolar macrophages and type II cells actively trans- port and accumulate taurine, suggesting that taurine might exert a protective effect against oxidant injury to these pneumocytes.

We conclude from these findings that cyclophospha- mide-induced early lung injury is associated with acute inflammatory reactions characterized by the secretion of lysosomal enzymes, free radical formation, increased levels of biomarkers in lung lavage fluid and decreased levels of antioxidants. Pretreatment with taurine plus niacin prevented cyclophosphamide-induced early lung derangements including oxidative damage. Taken to- gether with our results, it is therefore evident that the protective effect of taurine and niacin is related to the antiinflammatory, antioxidant, membrane-stabilizing property and ability to prevent epithelial damage. This scheme is consistent with the proposed antifibrotic role of these compounds.

Acknowledgements

The financial assistance to NVN by CSIR, New Delhi is gratefully acknowledged. The authors would also like to thank Dr. K.V. Raghavan, Director, Cen- tral Leather Research Institute, Madras, India, for his interest in publishing this work.

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