The Influence of Histamine H1-Receptor on Liver Functions in Immunized Rabbits

Saudi Journal of Biological Sciences (2011) 18, 411–418

King Saud University Saudi Journal of Biological Sciences

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE The Inﬂuence of histamine H1-receptor on liver functions in immunized rabbits

Trivendra Tripathi a,d,*, Mohammad Shahid b,*, Haris M. Khan b, Rahat Ali Khan c, Mashiatullah Siddiqui a, Abbas Ali Mahdi e a Department of Biochemistry, Faculty of Medicine, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh 202 002, UP, India b Department of Microbiology, Faculty of Medicine, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh 202 002, UP, India c Department of Pharmacology, Faculty of Medicine, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh 202 002, UP, India d Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA e Department of Biochemistry, Chhatrapati Shahuji Maharaj Medical University, UP, Lucknow, India

Received 21 September 2010; revised 17 July 2011; accepted 29 July 2011 Available online 5 August 2011

KEYWORDS Abstract This study was designed to investigate the functional roles of histamine and histamine Histamine; H1-receptor agonist and antagonist in the development of liver function impairment in immunized HTMT; rabbits. The study comprised of six groups containing 18 rabbits each. Group III–VI received his- Agonist; tamine (100 lg/kg, s.c.), H1R-agonist (HTMT, 10 lg/kg, s.c.), H1R-antagonist (pheniramine, Antagonist; 10 mg/kg, i.m.), and H1R-antagonist (pheniramine, 10 mg/kg, i.m.) plus histamine (100 lg/kg, ALT; s.c.), respectively, b.i.d. for 10 days. Group I (negative control) and group II (positive control) AST; received sterile distilled water intramuscularly b.i.d. for 10 days. Groups II–VI were immunized ALP; on day 3 with intravenous injection of SRBC (1 · 109 cells/ml). Blood samples were collected on

* Corresponding authors. Current Address: Department of Cell Biology and Anatomy, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX-76107, USA. Tel.: +1 817 908 8782 (T. Tripathi). E-mail addresses: [email protected], tripathiamu@gmail. com (T. Tripathi), [email protected], shahidsahar@ yahoo.co.in (M. Shahid).

Peer review under responsibility of King Saud University. doi:10.1016/j.sjbs.2011.07.002

Production and hosting by Elsevier 412 T. Tripathi et al.

Bilirubin; pre-immunization day 0, as well as on days 7-, 14-, 21-, 28-, and 58-post-immunization. Biochemical Rabbit parameters AST, ALT, alkaline phosphatase and bilirubin [total bilirubin (TB), direct bilirubin (DB), and indirect bilirubin (IB)] were determined. On each experimental day, the mean values of serum enzymes and bilirubin in group I and group II showed no significant changes while in group III, IV, V, and VI, these enzymes and bilirubin levels showed significant changes (p < 0.05), when compared with their experimental values within the group. The levels of serum enzymes and bilirubin showed significant difference (p < 0.05) in group III, IV, V, and VI on each experimental day, when compared with the corresponding values of each other, and also compared with the corresponding values of group I and II. Histamine, HTMT, pheniramine, and combination of histamine + pheniramine cause hepatic function impairment in terms of altered serum enzymes and bilirubin levels. The present findings suggest that HTMT causes moderate liver function impairment while others show mild impairment. ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction Recently, it has been demonstrated that histamine receptors (HR) on induction via their specific agonist can stimulate Histamine H1 receptor (H1R) expression has been demon- hypertrophy and hyperplasia of respiratory tract epithelium, strated to change in the disorders such as allergic rhinitis, and suggested HR agonists’ role akin to growth stimulating autoimmune myocarditis, rheumatoid arthritis and athero- factor (Dilkash et al., 2010). sclerosis (Tripathi et al., 2010a). Histamine H1R antagonists Liver is a vital organ for the biotransformation of xenobiot- have a long history of clinical efficacy in a variety of allergic ics and therefore is also exposed to drug toxicity including his- disorders. Several studies have reported changes in the tamine and HR ligands (Tripathi et al., 2010b). Histamine expression of H1R in pathological situations. Increased acting via histamine H2R pathway is believed to protect the expression of H1R was observed in the nasal mucosa of pa- liver against early alcohol-induced liver injury in rats (Hornyak tients with allergic rhinitis and in cultured aortic intimal et al., 2003) and also plays a role in the treatment of endotoxin- smooth muscle cells of the patients suffering from atheroscle- induced hepatic injury and related inflammatory disorders rosis (De Backer et al., 1998). Also, it has been documented (Masaki et al., 2005). Interestingly, different drugs acting that up-regulation of the H1R expression might have a role through the same receptors show different roles on the same in the initiation and progression of cardiovascular disease. In- biological process e.g., H2R antagonists like cimetidine and creased H1R expression has been reported in the inflamed ranitidine have inhibitory effect on liver regeneration but famo- joints of rheumatoid arthritis patients and in the heart of tidine has no such effect (Kanashima and Kobayashi, 1989). It mice with autoimmune myocarditis (De Backer et al., has also been demonstrated that modest inflammation predis- 1998). Moreover, it has been demonstrated that the exacerba- poses liver to ranitidine toxicity in rats and suggested a role tion of asthmatic symptoms experienced by some women dur- for the inflammation in idiosyncratic reactions to ranitidine ing pregnancy is due to the enhanced expression of H1R in (Luyendyk et al., 2003). nasal epithelial cells on induction by female sex hormones In contrast to studies on histamine and HR agonists and (De Backer et al., 1998). antagonists in various pathological conditions the data on H1R antihistamines, known as histamine H1R blockers or H1R agonists and antagonists on hepatic functions are ele- antagonists, are specific for the H1-receptor (De Backer et al., mentary in the existing literature. This prompted us to look 1998). Some H1R antagonists inhibit transmission through for their roles in hepatotoxicity (if any) in albino rabbits by the muscarinic, a-adrenergic, and serotonin receptors and also using liver function test. through ion channels. The H1R-antihistamines have been reclassified as inverse agonists, rather than as H1R antagonists, 2. Materials and methods which is consonant with an increased understanding of their molecular pharmacologic features (Simons, 2004). More than 2.1. Experimental design 40 H1R antagonists are available worldwide, indeed these H1R ligands are among the most widely used of all medications. To evaluate the serum level of liver enzymes and bilirubin, 108 However, the H1R antagonists (astemizole and terfenadine), (54 male and 54 female) New Zealand adult healthy albino which are associated with cardiac toxic effects, are no longer ap- rabbits of either sex weighing 1.0–1.5 kg were randomized proved for use (Simons, 2004). Both health care professionals equally into six treatment groups. Each group contained 18 and consumers assume that all the approved H1R antagonists (9 male and 9 female) rabbits. All groups were immunized with have been shown to be efficacious and safe, but several medica- sheep red blood cells (SRBC). Group I (negative control) and tions in this class have not been optimally studied in random- group II (positive control) received vehicle (sterile distilled ized, double-blind, controlled trials (Simons, 2004). water, 1 ml/kg · b.i.d.) while groups III, IV, V, and VI were First and second-generation H1R antagonists are reported treated with histamine, H1R agonist, H1R antagonist and to cause adverse effects, the mechanisms for which are incom- H1R antagonist + histamine, respectively. pletely understood. These effects include fixed-drug eruption, They were housed in a well maintained animal facility at photosensitivity, urticaria, fever, elevation of liver enzymes Central Animal House, J. N. Medical College, Aligarh Muslim and hepatitis, and agranulocytosis (Simons, 2004). University, Aligarh, in the Bioresources unit under 12 h light/ The Influence of histamine H1-receptor on liver functions in immunized rabbits 413 dark cycle, temperature (22 ± 2 C) and were allowed free bin (TB), direct bilirubin (DB), and indirect bilirubin (ID)] were access to standard laboratory diet, including green vegetables, determined using an automatic analyzer (Transasia XL 300, and tap water until experimentation. All the studies were Germany). carried out during the light cycle and were approved by the Institutional Animal Ethical Committee. 2.7. Statistical analyses

2.2. Drugs Data were summarized as Mean ± SD. Groups were compared by using repeated measures (subjects within groups) Histamine dihydrochloride was purchased from Himedia Lab- two way analysis of variance (ANOVA) followed by New- oratories Pvt. Limited, India. man–Keuls post hoc test. A two-tailed (a = 2) probability H1R agonist (histamine trifluoro-methyl toluidide (HTMT)- p < 0.05 was considered to be statistically significant. Analyses dimaleate) was kindly donated by Tocris Bioscience, Tocris were performed on SPSS for windows (version 12.0, Inc., Cookson Ltd., United Kingdom; H1R antagonist [Avil (phe- Chicago, IL). niramine maleate)] in injection I.P. from Unimark Remedies, India. 3. Results 2.3. Dosage regimen The changes in the mean values of serum level of serum enzymes (ALT, AST, and ALP) and bilirubin (TB, DB, and Histamine dihydrochloride 100 lg/kg and H1R agonist IB) in group I–VI are shown in Figs. 1–6, respectively. (HTMT-dimaleate) 10 lg/kg were administered through sub- On each experimental day, the mean values of serum level cutaneous (s.c.) route, and H1R antagonist (pheniramine of serum enzymes (ALT, AST, and ALP) and bilirubin (TB, maleate) 10 mg/kg was administered through intramuscular DB, and IB) in group I (negative control) and group II (posi- (i.m.) route; twice in a day [12 hourly (8 am and 8 pm)] for tive control) showed no significant changes, when compared 10 days (starting from 3 days prior to immunization until with the experimental values of serum enzymes and bilirubin 7 days after immunization). All doses referred to the weight within the group, and also compared with the corresponding of the salts used. values of each other. In groups III–VI, these enzymes and bilirubin levels showed changes on each experimental day, 2.4. Antigen when compared with the experimental values of serum enzymes and bilirubin within the group. Sheep blood diluted 1:1 in sterile Alsevier’s solution was Furthermore, the levels of serum enzymes and bilirubin obtained from the Department of Microbiology, J. N. Medical showed significant difference (p < 0.05) in groups III–VI on College, A.M.U., Aligarh, and washed with PBS (pH 7.4) each experimental day, when compared with the correspond- thrice by centrifugation. The cell suspensions were adjusted ing values of each other, and also compared with the corre- to the desired concentration in terms of hemoglobin, lysis of sponding values of groups I and II. a 1% SRBC suspension (2 · 108 cells/ml) with 14 volumes of 0.1% Na2CO3 develops an optical density of 0.135 at 541 nm in a spectrophotometer (Systronics, UV–visible double beam 4. Discussion spectrophotometer-2101, India) (Tripathi et al., 2010c–g). Fi- nally the concentration was adjusted to 5% (1 · 109 cells/ml) It has been demonstrated that liver of rabbits was markedly in PBS for immunization before use. damaged by histamine and HR (H1R, H2R, H3R, and H4R) agonist (Tripathi et al., 2010b). The damage caused by 2.5. Immunization of rabbits short-term exposure to histamine and HR agonist appeared quite specific for each of the agonist (Tripathi et al., 2010b). In general, increased incidence of hepatocyte polyploidy (bi- All the rabbits in the study groups (II–VI) were immunized on and multinucleated), frequent occurrence of Kupffer cells day 3 intravenously (i.v.) with 1 ml of 5% (1 · 109 cells/ml) (KCs), variable grades of necrosis, congestion, and lympho- sheep red blood cells (SRBC) in PBS. cytic infiltration constituted the hallmark of injuries were observed in histamine and HR agonist treated rabbits 2.6. Biochemical analyses (Tripathi et al., 2010b). Moreover, it has been shown that the elevation of endogenous histamine by amodiaquine (a his- To determine the biochemical levels for liver function, blood tamine N methyltransferase inhibitor) plays a protective role samples were collected from rabbits through the marginal ear through the regulation of TNF-a production in endotoxin- veins prior to immunization (day 0), as well as on days 7-, induced hepatic injury in mice (Yokoyama et al., 2007). 14-, 21-, 28-, and 58-post-I. Blood samples were kept at room Imoto et al. (1985) suggesting that histamine by H1R plays temperature for 2 h and then at 4 C overnight. Blood samples an important role in liver function. In addition to this, we have were centrifuged for 10 min at 580·g, and serum was separated designed our study to explore histamine and H1R agonist and and heated at 56 C for 30 min to inactivate the complement antagonist induced liver functions in immunized [with SRBC, proteins and stored in aliquots containing sodium azide as a a T cell-dependent test antigen (Tripathi et al., 2010c–g)] and preservative at À20 C. Serum levels of liver enzymes, including non-immunized rabbits. aspartate aminotransferase (AST), alanine aminotransferase The abnormalities in liver function tests (LFTs) are ele- (ALT), alkaline phosphatase (ALP), and bilirubin [total biliru- vated levels of static biochemical tests, including liver enzymes 414 T. Tripathi et al.

Days (pre and post)-immunization 0 (pre-I) 7 (post-I) 14 (post-I) 21 (post-I) 28 (post-I) 58 (post-I) 50

Serum ALT Level (IU/L) Level ALT Serum 10

5 0 Group I : Negative Group II : Positive Group III : Histamine Group IV : H1R- Group V : H1R- Group VI : H1R- Control Control Agonist Antagonist Antagonist + Histamine Experimental Groups

Figure 1 Effects of histamine and histamine H1-receptor agonist and antagonist on serum ALT levels. The results demonstrate mean ± s.d. of three experiments each with six rabbits. Two-way ANOVA followed by Newman–Keuls post hoc test revealed that the effect of treatments (F = 1163.937, DF = 5102; p < 0.01) and days (F = 237.363, DF = 5510; p < 0.01) on SRBC were statistically signiﬁcant. The interaction (treatments · days) effect of (F = 285.644, DF = 25,510; p < 0.01) these on SRBC was also found to be signiﬁcant.

Days (pre and post)-immunization 0 (pre-I) 7 (post-I) 14 (post-I) 21 (post-I) 28 (post-I) 58 (post-I) 40

10 Serum AST Level (IU/L) Level AST Serum

0 Group I : Negative Group II : Positive Group III : Histamine Group IV : H1R- Group V : H1R- Group VI : H1R- Control Control Agonist Antagonist Antagonist + Histamine Experimental Groups

Figure 2 Effects of histamine and histamine H1-receptor agonist and antagonist on serum AST levels. The results demonstrate mean ± s.d. of three experiments each with six rabbits. Two-way ANOVA followed by Newman–Keuls post hoc test revealed that the effect of treatments (F = 482.721, DF = 5102; p < 0.01) and days (F = 122.566, DF = 5510; p < 0.01) on SRBC were statistically signiﬁcant. The interaction (treatments · days) effect of (F = 177.821, DF = 25,510; p < 0.01) these on SRBC was also found to be signiﬁcant.

(ALT, AST, and alkaline phosphatase) and bilirubin, which et al., 2005; Thapa and Walia, 2007). The biotransformation are the biochemical parameters of liver damage (Masaki of xenobiotics takes place in liver (Maciejewska-Paszek et al., The Inﬂuence of histamine H1-receptor on liver functions in immunized rabbits 415

Days (pre and post)-immunization 0 (pre-I) 7 (post-I) 14 (post-I) 21 (post-I) 28 (post-I) 58 (post-I) 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5

Serum Alkaline phosphatase Level (IU/L) Level phosphatase Alkaline Serum 0 Group I : Negative Group II : Positive Group III : Histamine Group IV : H1R- Group V : H1R- Group VI : H1R- Control Control Agonist Antagonist Antagonist + Histamine Experimental Groups

Figure 3 Effects of histamine and histamine H1-receptor agonist and antagonist on serum alkaline phosphatase (ALP) levels. The results demonstrate mean ± s.d. of three experiments each with six rabbits. Two-way ANOVA followed by Newman–Keuls post hoc test revealed that the effect of treatments (F = 696.678, DF = 5102; p < 0.01) and days (F = 742.821, DF = 5510; p < 0.01) on SRBC were statistically signiﬁcant. The interaction (treatments · days) effect of (F = 276.182, DF = 25,510; p < 0.01) these on SRBC was also found to be signiﬁcant.

Days (pre and post)-immunization 0 (pre-I) 7 (post-I) 14 (post-I) 21 (post-I) 28 (post-I) 58 (post-I) 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 Serum Total bilirubin Level (mg/dl) Level bilirubin Total Serum 0.05 0 Group I : Negative Group II : Positive Group III : Histamine Group IV : H1R- Group V : H1R- Group VI : H1R- Control Control Agonist Antagonist Antagonist + Histamine Experimental Groups

Figure 4 Effects of histamine and histamine H1-receptor agonist and antagonist on serum total bilirubin (TB) levels. The results demonstrate mean ± s.d. of three experiments each with six rabbits. Two-way ANOVA followed by Newman–Keuls post hoc test revealed that the effect of treatments (F = 775.278, DF = 5102; p < 0.01) and days (F = 199.418, DF = 5510; p < 0.01) on SRBC were statistically signiﬁcant. The interaction (treatments · days) effect of (F = 134.927, DF = 25,510; p < 0.01) these on SRBC was also found to be signiﬁcant.

2007), therefore, the LFTs are the most common blood tests The most common abnormalities of LFTs in an asymptomatic required by clinical practitioners for examining liver damage. person are increased ALT and AST. The upper normal limits 416 T. Tripathi et al.

Days (pre and post)-immunization 0 (pre-I) 7 (post-I) 14 (post-I) 21 (post-I) 28 (post-I) 58 (post-I) 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 Serum Direct bilirubin Level (mg/dl) Level bilirubin Direct Serum 0.05 0 Group I : Negative Group II : Positive Group III : Histamine Group IV : H1R- Group V : H1R- Group VI : H1R- Control Control Agonist Antagonist Antagonist + Histamine Experimental Groups

Figure 5 Effects of histamine and histamine H1-receptor agonist and antagonist on serum direct bilirubin (DB) levels. The results demonstrate mean ± s.d. of three experiments each with six rabbits. Two-way ANOVA followed by Newman–Keuls post hoc test revealed that the effect of treatments (F = 469.121, DF = 5102; p < 0.01) and days (F = 142.24, DF = 5510; p < 0.01) on SRBC were statistically signiﬁcant. The interaction (treatments · days) effect of (F = 111.402, DF = 25,510; p < 0.01) these on SRBC was also found to be signiﬁcant.

Days (pre and post)-immunization 0 (pre-I) 7 (post-I) 14 (post-I) 21 (post-I) 28 (post-I) 58 (post-I) 0.275 0.25 0.225 0.2 0.175 0.15 0.125 0.1 0.075 0.05

Serum Indirect bilirubin Level (mg/dl) Level bilirubin Indirect Serum 0.025 0 Group I : Negative Group II : Positive Group III : Histamine Group IV : H1R- Group V : H1R- Group VI : H1R- Control Control Agonist Antagonist Antagonist + Histamine Experimental Groups

Figure 6 Effects of histamine and histamine H1-receptor agonist and antagonist on serum indirect bilirubin (IB) levels. The results demonstrate mean ± s.d. of three experiments each with six rabbits. Two-way ANOVA followed by Newman–Keuls post hoc test revealed that the effect of treatments (F = 71.109, DF = 5102; p < 0.01) and days (F = 6.737, DF = 5510; p < 0.01) on SRBC were statistically significant. The interaction (treatments · days) effect of (F = 7.876, DF = 25,510; p < 0.01) these on SRBC was also found to be significant. of ALT and AST differ from lab to lab (Giannini et al., 2005). may be raised in patients with muscle injury (Hasan and AST and ALT reflect liver degeneration. Both AST and ALT Owyed, 2003). In most liver diseases, AST/ALT ratio is <1. The Influence of histamine H1-receptor on liver functions in immunized rabbits 417

However, in alcoholic liver disease (alcoholic steatosis, It is therefore concluded that histamine, HTMT (H1R alcoholic hepatitis, and cirrhosis), AST/ALT is >1, and is agonist), pheniramine (H1R antagonist), and combination of usually >2 (Sorbi et al., 1999). Mild to moderate elevations pheniramine+histamine cause hepatic function impairment in serum ALT and AST levels may be observed in virtually in terms of altered serum enzymes and bilirubin levels. Thus, all liver diseases, and thus are generally nonspecific (Mathiesen the present findings suggest that HTMT causes moderate liver et al., 1999). ALT and AST levels exceeding 15 times the upper function impairment while others show mild impairment war- limit of normal are usually found in a limited number of con- ranting further long-term studies. This study would definitely ditions (i.e. drugs, acute viral hepatitis, ischemic hepatitis, stimulate active discussions about the effects of histamine autoimmune hepatitis, common bile duct stones and muscle in- and its HR agonists and antagonists on hepatic functions. jury) (Fortson et al., 1985; Johnson et al., 1995; Mathiesen To the best of our knowledge, this is the first report describing et al., 1999). ALT and AST are sensitive markers of hepatocel- a comparative study of histamine and H1R agonist and antag- lular injury but they lack specificity as they are also present in onist on liver function in immunized and non-immunized muscle (cardiac and skeletal), kidney and red blood cells (Yap rabbits. and Aw, 2010). Serum alkaline phosphatase (ALP) activity may derive from liver, bone, intestine, and placenta. Various Acknowledgments hepatobiliary (such as drugs, bile duct obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, liver metasta- T. Tripathi acknowledges University Grants Commission sis, infiltrative liver disease, viral hepatitis, alcoholic hepatitis, (UGC), New Delhi, India for providing UGC Fellowship and cirrhosis) and non-hepatic (bone disease, pregnancy, [UGC letter DON F. 19-33/2006 (CU)]. The authors wish to chronic renal failure, congestive heart failure, childhood thank Tocris Bioscience, Tocris Cookson Ltd. (United growth, and malignancy) causes are related with raised ALP. Kingdom) for kindly donating histamine receptor’s agonists However, low serum ALP in patients with biochemical evi- and antagonists. dence of liver dysfunction may be a clue to Wilson’s disease. Also, low ALP may be encountered in hypothyroidism, zinc References deficiency, pernicious anemia, and congenital hypophosphate- mia (Hasan and Owyed, 2003). The results of this study De Backer, M.D., Loonen, I., Verhasselt, P., Neefs, J.M., Luyten, revealed that groups III–VI show impaired serum levels of W.H.M.L., 1998. Structure of the human histamine H1 receptor ALT, AST, and ALP. This study shows moderate alterations gene. Biochem. J. 335 (Pt 3), 663–670. in serum levels of ALT, AST, and ALP in group IV (HTMT) Dilkash, N.A., Tripathi, T., Khan, A.A., Shahid, M., Khan, while group III (histamine), group V (pheniramine), and group H.M., Siddiqui, M.U., Khan, R.A., 2010. Effects of histamine VI (pheniramine+histamine) show mild impairment. Thus, H1R-H4R-agonist (histamine trifluoro-methyl toluidide, amth- serum levels of ALT and AST impairment represent liver dam- amine, R-(À)-a-methyl histamine and clobenpropit, respectively) on the airway epithelium of rabbits. Asian Pacific J. Trop. age in the order: HTMT > pheniramine > pheniramine+his- Med. 3 (5), 367–370. tamine > histamine, when compared with positive control and Fortson, W.C., Tedesco, F.J., Starnes, E.C., Shaw, C.T., 1985. Marked negative control. elevation of serum transaminase activity associated with extrahe- Furthermore, serum bilirubin is a mixture of a, b, c, and d patic biliary tract disease. J. Clin. Gastroenterol. 7 (6), 502–505. fragments which are unconjugated, singly conjugated, doubly Friedman, S.F., Martin, P., Munoz, J.S., 2003. Laboratory evaluation conjugated, and covalently bound to albumin, respectively of the patient with liver disease. In: Boyer, Zakim (Eds.), (Yap and Aw, 2010). Although d bilirubin measurement is Hepatology, a textbook of liver disease. Philedelphia: Saunders available, it has not gained wider utility. In most cases a total publication, pp. 661–709. bilirubin (TB) assay suffices for LFT, but fractionation may Giannini, E.G., Testa, R., Savarino, V., 2005. Liver enzyme alteration: be required in isolated increases in bilirubin and neonatal a guide for clinicians. Can. Med. Assoc. J. 172 (3), 367–379. Hasan, F.A.M., Owyed, S., 2003. 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DB, and IB in groups III-VI show liver function impairment Maciejewska-Paszek, I., Pawowska-Go´ral, K., Kostrzewski, M., in the order: histamine > HTMT > pheniramine+hista- Kurzeja, E., Wardas, M., Rzepecka-Stojko, A., 2007. The influence mine > pheniramine, when compared with positive control of small doses of paracetamol on rabbit liver. Exp. Toxicol. Pathol. and negative control. 59 (2), 139–141. 418 T. Tripathi et al.

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