Article
Effects of formaldehyde inhalation on humoral immunity and protective effect of Nigella sativa oil: An experimental study
Toxicology and Industrial Health 1–6 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714566294 tih.sagepub.com
Hilal Irmak Sapmaz1, Mustafa Sarsılmaz2, ¨ getu¨rk4, Ufuk Tas¸ 5 Ahmet Go¨dekmerdan3, Murat O 1 and Evren Ko¨se Abstract Aim: This study was carried out to determine the effects of formaldehyde (FA) inhalation on the humoral immunity of rats and the protective effect of Nigella sativa (NS) oil. Materials and Methods: The rats (n ¼ 33) were divided into five groups, with five animals in the control group (FA-free air) and seven in the other four groups. Group FA1 was exposed to FA (5 ppm), group FA þ NS1 was treated with NS and exposed to FA (5 ppm), group FA2 was exposed to FA (10 ppm), and group FA þ NS2 was treated with NS and exposed to FA (10 ppm). At the end of a 4-week study period, blood samples were collected. Enzyme-linked immunosorbent assay was used to determine the levels of serum total immunoglobulin A (IgA), total immunoglobulin M (IgM), total immunoglobulin G (IgG), and complement 3 (C3). Results: FA inhalation significantly increased serum IgA, IgM, and C3 levels and decreased serum IgG levels compared with the control group. NS administration decreased serum IgA, IgM, and C3 levels, which were induced by FA inhalation. Conclusion: FA inhalation significantly increased acute antibody responses and C3 levels in a dose-dependent manner compared with the control group. FA inhalation decreased the secondary immune response compared with the control group. Levels of acute antibody responses and complement following exposure to FA inhalation returned to normal following treatment with NS (immunoregulatory effect). However, NS did not affect the secondary immune response. Keywords Formaldehyde, humoral immunity, Nigella sativa oil, total immunoglobulin, ELISA
Introduction
1
Formaldehyde (FA) is a carcinogenic substance with harmful effects on human health (Kerns et al., 1983; Wilmer et al., 1989). FA is a colorless, reactive agent, which is easily soluble in water. It is an endogenous metabolic product (Cheney and Collins, 1995; Smith, 1992). FA is widely used in dyes, plastics, textiles, and leather industries and in the manufacturing of cleaning and cosmetic products, some wooden products, varnish, paper, ink, adhesives, and sugar. FA is also used as a preservative in some foods and as an additive in some drugs. (ATSDR, 1999; Bernstein et al., 1984; Feron et al., 1991; Heck et al., 1990).
Corresponding author: Mustafa Sarsılmaz, Department of Anatomy, Faculty of Medicine, Sifa University, Izmir, Turkey. Email: [email protected]
Department of Anatomy, Faculty of Medicine, Inonu University, Malatya, Turkey 2 Department of Anatomy, Faculty of Medicine, Sifa University, Izmir, Turkey 3 Department of Microbiology, Faculty of Medicine, Yildirim Beyazit University, Ankara, Turkey 4 Department of Anatomy, Faculty of Medicine, Firat University, Elazig, Turkey 5 Department of Anatomy, Faculty of Medicine, Gaziosmanpas¸ a University, Tokat, Turkey
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Experimental studies in animals indicated that FA may damage the nervous and reproductive systems (Ozen et al., 2002; Sarsilmaz et al., 2007; Songur et al., 2010; Zararsiz et al., 2007a). Other studies reported that FA has mutagenic and carcinogenic properties and that it may harm the kidneys (Stroup et al., 1986; Yager et al., 1986; Zararsiz et al., 2007b). Immunoglobulin G (IgG) comprises the largest part of normal human serum immunoglobulin. IgG may reach very high levels, especially during the secondary immune response. Immunoglobulin M (IgM) is the main antibody of the early primary immune response. Immunoglobulin A (IgA) is the main immunoglobulin in secretions. The complement (C) system has an important role in the body’s defense mechanisms against microorganisms, with C3 the main component of the system (Abbas and Lichtman, 2004). Foreign agents, especially toxic chemicals, can seriously affect the immune system of the body. Oral administration of FA reduced the levels of IgG and IgM in rats, depending on the dose (Vargova´ et al., 1993). Occupational FA exposure decreased white blood cell counts in the blood (Kuo et al., 1997). Nigella sativa (NS) is an annual herbaceous plant, which belongs to the Ranunculaceae family. The value of NS is well known in local folk medicine, and they can be found in herbalist shops in many countries (Phillips, 1992). NS seeds were reported to have antioxidant, antimalarial, antihyperglycemic, radioprotective, and immunomodulator properties (Assayed, 2010; Benhaddou-Andaloussi et al., 2008; Boskabady et al., 2011; Okeola et al., 2011). The neuronprotective effect of NS was demonstrated in experimental animal models (Kanter et al., 2006). The aim of this study was to investigate the possible harmful effects of FA on humoral immunity. In addition, the possible protective properties of NS oil against these adverse effects were investigated.
Materials and methods Animals and treatments
lighting (07.00–19.00 h). All the rats were allowed to acclimatize for a week before the experimentation. The rats were divided into five groups. Clean air was provided in an FA-free environment for 4 weeks to the rats in the control group (n ¼ 5). The other 28 rats were divided into four study groups. Group FA1 was exposed to FA (5 ppm/8 h/day) for 4 weeks (n ¼ 7), group FA þ NS1 was treated with NS oil (1 ml/kg/day by gavage) and exposed to FA (5 ppm) inhalation for 4 weeks (n ¼ 7), group FA2 was exposed to FA (10 ppm/8 h/day), and group FA þ NS2 was treated with NS oil (1 ml/kg/day by gavage) and exposed to FA (10 ppm) inhalation for 4 weeks (n ¼ 7). The concentrations of FA were those described by Ozen et al. (2005), and the FA exposure periods were those described by Ozen et al. (2002) and Sarsilmaz et al. (2007). The dose of NS was based on a previous study (Assayed, 2010). The rats inhaled FA 5 days per week at regular intervals in glass cages through two holes that were made in each cage to allow the entry and exit of air. During non-FA exposure periods, the rats were taken from their glass cages and returned to their own cages where they received standard laboratory chow (supplied from Elazig Feed Plant, Elazig, Turkey) and tap water ad libitum.
Production of FA and dose measurement Paraformaldehyde (Merck KGaA, 64271 Darmstadt, Germany) was heated at a temperature of 35–40 C and depolymerized (Chang et al., 1981) to obtain FA gas. Gaseous FA was pumped into the glass cages with the help of a pumping system, according to the exposure period and FA concentration. The FA concentration in the glass cages was continually monitored with an FA monitor (catalog no: ZDL-300, Environmental Sensors Co., Boca Raton, Florida, USA), as recommended by the Occupational Safety and Health Administration.
Sample collection and immunological study
Adult male Sprague Dawley rats (weighing 270–300 g, n ¼ 33) were obtained from the Experimental Research Unit, Firat University, Elazig, Turkey. The Local Ethics Committee for Animal Research approved the experimental protocol for this study, and all animals received humane care in compliance with the European Convention on Animal Care. The rats were housed in cages in an air-conditioned room with automatically regulated temperature (22 + 1 C) and
At the end of the study period, all the animals were killed by decapitation, and blood samples were collected in routine biochemical test tubes. The serum was separated by centrifugation and stored at 80 C until the total Igs and C3 titer levels were measured. Micro-enzyme-linked immunosorbent assay (ELISA) kits from the Kamiya Biomedical Company (Seattle, Washington, USA), plus a double antibody sandwich ELISA protocol and ELISA reader (ELx800 BioTek, Winooski, Vermont, USA), were
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Table 1. The levels of total IgA, IgM, IgG, and C3 (mean + SEM). Parameters
Control (n ¼ 5)
FA1 (n ¼ 7)
FA2 (n ¼ 7)
FA þ NS1 (n ¼ 7)
FA þ NS2 (n ¼ 7)
IgA (ng/ml)
59,411.58 + 3769.05 10,572.10 + 401.76 72,680.16 + 13715.65 16,421.98 + 610.53
107,754.10 + 9768.07a 17,685.13 + 849.52a 33,186.97 + 9454.43a 18,267.14 + 292.20a
109,556.23 + 8452.60a 20,486.19 + 1045.85a 32,424.00 + 3205.56a 19,198.73 + 1094.12a
61,873.96 + 5,081.54b 10,743.76 + 595.21b 37,124.31 + 4915.13a 16,591.29 + 141.23b
73,354.39 + 1985.21a,c 10,458.51 + 1160.19c 29,415.94 + 2973.03a 17,292.86 + 153.42
IgM (ng/ml) IgG (ng/ml) C3 (ng/ml)
IgA: immunoglobulin A; IgM: immunoglobulin M; IgG: immunoglobulin G; C3: complement 3; FA: formaldehyde; NS: Nigella sativa. a p < 0.05: compared with control. b p < 0.05: compared with FA1. c p < 0.05: compared with FA2.
used to determine the titer levels of total IgA, IgM, IgG, and C3 in the serum.
Statistical analysis A Kruskal–Wallis test was used because the data did not show a normal distribution. For multiple comparisons, a Mann–Whitney U test was used. Quantitative data were expressed as the mean + standard error of the mean. The level of significance was set at p < 0.05.
Results Total IgA and IgM levels increased in the FA-exposed groups (dose dependent) when compared with the control group (p < 0.05). Total IgA levels also increased significantly in the FA þ NS2 group compared with the control group (p < 0.05). When the FA groups were compared with the FA þ NS groups, there was a significant decrease in total IgA and IgM levels in the FA þ NS1 group compared with those in the FA1 group (p < 0.05). Similarly, these Igs were decreased in the FA þ NS2 group compared with those in the FA2 group (p < 0.05; Table 1; Figures 1 and 2). Based on total IgG levels, the secondary immune response decreased in all the FA inhalation groups when compared with the control group (p < 0.05). The administration of NS did not change the IgG levels in the FA þ NS1 and FA þ NS2 groups compared with the FA groups (Table 1; Figure 3). Comparison of the C3 levels of the groups exposed only to FA with those of the control group revealed a significant increase in the former (p < 0.05). Furthermore, the C3 levels of the FA þ NS1 group decreased compared with those of the FA1 group (p < 0.05).
Figure 1. The levels of the serum total IgA of all groups. a p < 0.05: compared with control; bp < 0.05: compared with FA1; and cp < 0.05: compared with FA2. IgA: immunoglobulin A; FA: formaldehyde; NS: Nigella sativa.
Figure 2. The levels of the serum total IgM of all groups. a p < 0.05: compared with control; bp < 0.05: compared with FA1; and cp < 0.05: compared with FA2. IgM: immunoglobulin M; FA: formaldehyde; NS: Nigella sativa.
However, there were no significant differences in C3 levels between the FA2 and FA þ NS2 groups (Table 1; Figure 4).
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Figure 3. The levels of the serum total IgG of all groups. a p < 0.05: compared with control. IgG: immunoglobulin G; NS: Nigella sativa.
Figure 4. The levels of the serum C3 of all groups. ap < 0.05: compared with control and bp < 0.05: compared with FA1. C3: complement 3; FA: formaldehyde; NS: Nigella sativa.
Discussion There are many experimental studies on the effects of different chemical agents on the immune system (Ayatollahi M, 2002; Bogadi-Sare et al., 2000; Lange et al., 1973; Mishra et al., 2006; Zhu et al., 2011). However, only a limited number of studies have investigated the effect of FA on the immune system (Baj et al., 1994; Dean et al., 1984; Kuo et al., 1997; Veraldi et al., 2006). One study reported that immune and hematopoietic systems may be affected if they are exposed to FA for a long time (Baj et al., 1994). However, another reported that bone marrow cellularity, lymphoid organ weights, and hematological parameters were unchanged in mice exposed to 15 ppm of FA for 21 days (Dean et al., 1984). Another study found that although FA binds to and is transported by albumin, the immune system is capable of producing an antibody against this complex (Coutinho et al., 1995). Several studies showed that serum IgA levels increased following exposure to various toxic agents
(Mishra et al., 2006; Zhu et al., 2011). However, some researchers reported that occupational exposure to certain toxic agents decreased serum IgA levels (BogadiSare et al., 2000; Lange et al., 1973; Oh et al., 2005). Mishra et al. (2006) found that serum IgA levels were increased in a group of lead-exposed workers compared to those of unexposed healthy controls. In an experimental study, levels of IgA increased in rats exposed to aluminum (Zhu et al., 2011). Several studies showed that IgA levels decreased in workers with occupational exposure to benzene or dioxin (BogadiSare et al., 2000; Lange et al., 1973; Oh et al., 2005). In our study, serum IgA levels increased significantly in response to FA inhalation. IgA is a mucosaprotecting antibody. Based on the current findings, we conclude that IgA levels increase in response to FA exposure. Terro´n et al. (2009) showed that IgA levels decreased in response to antioxidant administration. In this study, the administration of NS oil decreased total serum IgA levels, which increased following FA exposure. NS ameliorated the immunotoxic effects of low-dose FA, but NS did not ameliorate the immunotoxic effects of the high-dose FA exposure. In an experimental study, rats that received FA orally (20, 40, and 80 mg/kg) showed a dosedependent decrease in serum IgM levels (Vargova´ et al., 1993). Serum IgM levels decreased in response to exposure to some toxic agents (Oh et al., 2005; Zhu et al., 2011), whereas serum IgM levels increased in response to exposure to some other toxic agents (Bogadi-Sare et al., 2000; Lange et al., 1973). In our study, the IgM values of the FA groups were elevated compared with those of the control group. We assume that the increase in IgM values in this study may have been an acute response of the immune system to the exposure to FA. In an experimental study, the serum IgM levels of birds treated with melatonin increased (Terro´n et al., 2009). In our study, the administration of NS oil decreased the serum total IgM levels, which were elevated in response to FA exposure. The acute immune response (IgM) caused both by the low- and high-dose FA returned to normal levels following the NS treatment. Aluminum exposure has been shown to increase serum IgG levels in rats (Zhu et al., 2011). However, oral administration of FA decreased serum IgG levels in rats in a dose-dependent manner (Vargova´ et al., 1993). Several studies of benzene and dioxin reported a dose-dependent decrease in serum IgG values (Lange et al., 1973; Oh et al., 2005). Ayatollahi (2002) found a significant reduction in the IgG levels of humans with
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occupational exposure to lead. Similarly, we found a significant decline in the IgG levels of the rats exposed to FA compared with those of the control group. This result suggests that FA inhalation substantially suppressed the secondary immune antibody response (IgG). In another study, melatonin administration increased serum levels of IgG compared to their control values in birds (Terro´n et al., 2009). In accordance with our findings, the NS oil did not improve the serum IgG levels, which were depressed by FA exposure. Several studies showed that dioxin exposure can increase C levels (Mocarelli et al., 1986; Ott et al., 1994). On the other hand, rats exposed to aluminum had decreased levels of C3 and C4 (Zhu et al., 2011). We found a significant increase in the serum C3 levels of the FA groups compared with the control group. This suggests that there is an important humoral immune response to FA. The return of elevated C3 levels following FA exposure to normal values after the administration of the NS oil suggests that cells that produce C3 are not irrevocably damaged by FA (reversible effect). In another study of rats challenged with typhoid antigen, treatment with NS volatile oil reduced the serum antibody titer compared with the control animals (Islam et al., 2004). Likewise, in this study, FA exposure increased serum IgA and IgM levels. These antibodies were decreased in the FA þ NS group compared with those of the FA groups.
Conclusion The acute antibody response (IgA and IgM) and C3 levels in serum increased significantly in the FA groups (dose dependent) compared with the control group. Moreover, the secondary immune response (IgG) of the FA groups decreased compared with the control group. The elevated responses of C and acute antibody to FA, which is a foreign antigen for the organism, returned to normal levels following the NS treatment (immunoregulatory effect). However, NS had no effect on the secondary immune response. Long-term studies with larger numbers of animals and with doses closer to those observed in humans are required to determine the immunotoxic effects of FA, the protective effects of NS, and related cellular and humoral immune parameters. Authors’ Note This study was presented partially at Joint Meeting of Anatomical Societies, 19–22 May 2011, Bursa, Turkey, as a poster.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Dean JH, Lauer LD, House RV, et al. (1984) Studies of immune function and host resistance in B6C3F1 mice exposed to formaldehyde. Toxicology and Applied Pharmacology 72: 519–529. Feron VJ, Til HP, de Vrijer F, et al. (1991) Aldehydes: occurrence, carcinogenic potential, mechanism of action and risk assessment. Mutation Research 259: 363–385. Heck HD, Casanova M and Starr TB (1990) Formaldehyde toxicity—new understanding. Critical Reviews in Toxicology 20: 397–426. Islam SN, Begum P, Ahsan T, et al. (2004) Immunosuppressive and cytotoxic properties of Nigella sativa. Phytotherapy Research: PTR 18: 395–398. Kanter M, Coskun O, Kalaycı M, et al. (2006) Neuroprotective effects of Nigella sativa on experimental spinal cord injury in rats. Human & Experimental Toxicology 25: 127–133. Kerns WD, Pavkov KL, Donofrio DJ, et al. (1983) Carcinogenicity of formaldehyde in rats and mice after long-term inhalation exposure. Cancer Research 43: 4382–4392. Kuo H, Jian G, Chen C, et al. (1997) White blood cell count as an indicator of formaldehyde exposure. Bulletin of Environmental Contamination and Toxicology 59: 261–267. Lange A, Smolik R, Zatonski W, et al. (1973) Serum immunoglobulin levels in workers exposed to benzene, toluene and xylene. Internationales Archiv fu¨r Arbeitsmedizin 3: 37–44. Mishra KP, Chauhan UK and Naik S (2006) Effect of lead exposure on serum immunoglobulins and reactive nitrogen and oxygen intermediate. Human & Experimental Toxicology 25: 661–665. Mocarelli P, Marocchi A, Brambilla P, et al. (1986) Clinical laboratory manifestations of exposure to dioxin in children. A six-year study of the effects of an environmental disaster near Seveso, Italy. Journal of the American Medical Association 256: 2687–2695. Oh E, Lee E, Im H, et al. (2005) Evaluation of immuno- and reproductive toxicities and association between immunotoxicological and genotoxicological parameters in waste incineration workers. Toxicology 210: 65–80. Okeola VO, Adaramoye OA, Nneji CM, et al. (2011) Antimalarial and antioxidant activities of methanolic extract of Nigella sativa seeds (black cumin) in mice infected with plasmodium Yoelli nigeriensis. Parasitology Research 108: 1507–1512. Ott MG, Zober A and German C (1994) Laboratory results for selected target organs in 138 individuals occupationally exposed to TCDD. Chemosphere 29: 2423–2437. Ozen OA, Yaman M, Sarsilmaz M, et al. (2002) Testicular zinc, copper and iron concentrations in male rats exposed to subacute and subchronic formaldehyde gas inhalation. Journal of Trace Elements in Medicine and
Biology: Organ of the Society for Minerals and Trace Elements (GMS) 16: 119–122. Ozen OA, Akpolat N, Songur A, et al. (2005) Effect of formaldehyde inhalation on Hsp 70 in seminiferous tubules of rat testes: an immunohistochemical study. Toxicology and Industrial Health 21(9): 249–254. Phillips JD (1992) Medicinal plants. Biologist 39: 187–191. Sarsilmaz M, Kaplan S, Songur A, et al. (2007) Effects of postnatal formaldehyde exposure on pyramidal cell number, volume of cell layer in hippocampus and hemisphere in the rat: a stereological study. Brain Research 1145: 157–167. Smith AE (1992) Formaldehyde. Occupational Medicine 42: 83–88. Songur A, Ozen OA and Sarsilmaz M (2010) The toxic effects of formaldehyde on the nervous system. Reviews of Environmental Contamination and Toxicology 203: 105–118. Stroup NE, Blair A and Erikson GE (1986) Brain cancer and other causes of death in anatomists. Journal of the National Cancer Institute 77: 1217–1224. Terro´n MP, Delgado J, Paredes SD, et al. (2009) Effect of melatonin and tryptophan on humoral immunity in young and old ringdoves (Streptopelia risoria). Experimental Gerontology 44: 653–658. Vargova´ M, Wagnerova´ J, Lı´skova´ A, et al. (1993) Subacute immunotoxicity study of formaldehyde in male rats. Drug and Chemical Toxicology 16: 255–275. Veraldi A, Costantini AS, Bolejack V, et al. (2006) Immunotoxic effects of chemicals: a matrix for occupational and environmental epidemiological studies. American Journal of Industrial Medicine 49: 1046–1055. Wilmer JW, Woutersen RA, Appelman LM, et al. (1989) Subchronic (13-week) inhalation toxicity study of formaldehyde in male rats: 8-hour intermittent versus 8-hour continuous exposures. Toxicology Letters 47: 287–293. Yager JW, Cohn KL, Spear RC, et al. (1986) Sisterchromatid exchanges in lymphocytes of anatomy students exposed to formaldehyde-embalming solution. Mutation Research 174: 135–139. Zararsiz I, Kus I, Ogeturk M, et al. (2007a) Melatonin prevents formaldehyde-induced neurotoxicity in prefrontal cortex of rats: an immunohistochemical and biochemical study. Cell Biochemistry and Function 25: 413–418. Zararsiz I, Sarsilmaz M, Tas U, et al. (2007b) Protective effect of melatonin against formaldehyde-induced kidney damage in rats. Toxicology and Industrial Health 23: 573–579. Zhu Y, Xu J, Sun H, et al. (2011) Effects of aluminum exposure on the allergic responses and humoral immune function in rats. Biometals: An International Journal on the Role of Metal ions in Biology, Biochemistry, and Medicine 24: 973–977.
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Effects of formaldehyde inhalation on humoral immunity and protective effect of Nigella sativa oil: An experimental study
Toxicology and Industrial Health 1–6 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714566294 tih.sagepub.com
Hilal Irmak Sapmaz1, Mustafa Sarsılmaz2, ¨ getu¨rk4, Ufuk Tas¸ 5 Ahmet Go¨dekmerdan3, Murat O 1 and Evren Ko¨se Abstract Aim: This study was carried out to determine the effects of formaldehyde (FA) inhalation on the humoral immunity of rats and the protective effect of Nigella sativa (NS) oil. Materials and Methods: The rats (n ¼ 33) were divided into five groups, with five animals in the control group (FA-free air) and seven in the other four groups. Group FA1 was exposed to FA (5 ppm), group FA þ NS1 was treated with NS and exposed to FA (5 ppm), group FA2 was exposed to FA (10 ppm), and group FA þ NS2 was treated with NS and exposed to FA (10 ppm). At the end of a 4-week study period, blood samples were collected. Enzyme-linked immunosorbent assay was used to determine the levels of serum total immunoglobulin A (IgA), total immunoglobulin M (IgM), total immunoglobulin G (IgG), and complement 3 (C3). Results: FA inhalation significantly increased serum IgA, IgM, and C3 levels and decreased serum IgG levels compared with the control group. NS administration decreased serum IgA, IgM, and C3 levels, which were induced by FA inhalation. Conclusion: FA inhalation significantly increased acute antibody responses and C3 levels in a dose-dependent manner compared with the control group. FA inhalation decreased the secondary immune response compared with the control group. Levels of acute antibody responses and complement following exposure to FA inhalation returned to normal following treatment with NS (immunoregulatory effect). However, NS did not affect the secondary immune response. Keywords Formaldehyde, humoral immunity, Nigella sativa oil, total immunoglobulin, ELISA
Introduction
1
Formaldehyde (FA) is a carcinogenic substance with harmful effects on human health (Kerns et al., 1983; Wilmer et al., 1989). FA is a colorless, reactive agent, which is easily soluble in water. It is an endogenous metabolic product (Cheney and Collins, 1995; Smith, 1992). FA is widely used in dyes, plastics, textiles, and leather industries and in the manufacturing of cleaning and cosmetic products, some wooden products, varnish, paper, ink, adhesives, and sugar. FA is also used as a preservative in some foods and as an additive in some drugs. (ATSDR, 1999; Bernstein et al., 1984; Feron et al., 1991; Heck et al., 1990).
Corresponding author: Mustafa Sarsılmaz, Department of Anatomy, Faculty of Medicine, Sifa University, Izmir, Turkey. Email: [email protected]
Department of Anatomy, Faculty of Medicine, Inonu University, Malatya, Turkey 2 Department of Anatomy, Faculty of Medicine, Sifa University, Izmir, Turkey 3 Department of Microbiology, Faculty of Medicine, Yildirim Beyazit University, Ankara, Turkey 4 Department of Anatomy, Faculty of Medicine, Firat University, Elazig, Turkey 5 Department of Anatomy, Faculty of Medicine, Gaziosmanpas¸ a University, Tokat, Turkey
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Experimental studies in animals indicated that FA may damage the nervous and reproductive systems (Ozen et al., 2002; Sarsilmaz et al., 2007; Songur et al., 2010; Zararsiz et al., 2007a). Other studies reported that FA has mutagenic and carcinogenic properties and that it may harm the kidneys (Stroup et al., 1986; Yager et al., 1986; Zararsiz et al., 2007b). Immunoglobulin G (IgG) comprises the largest part of normal human serum immunoglobulin. IgG may reach very high levels, especially during the secondary immune response. Immunoglobulin M (IgM) is the main antibody of the early primary immune response. Immunoglobulin A (IgA) is the main immunoglobulin in secretions. The complement (C) system has an important role in the body’s defense mechanisms against microorganisms, with C3 the main component of the system (Abbas and Lichtman, 2004). Foreign agents, especially toxic chemicals, can seriously affect the immune system of the body. Oral administration of FA reduced the levels of IgG and IgM in rats, depending on the dose (Vargova´ et al., 1993). Occupational FA exposure decreased white blood cell counts in the blood (Kuo et al., 1997). Nigella sativa (NS) is an annual herbaceous plant, which belongs to the Ranunculaceae family. The value of NS is well known in local folk medicine, and they can be found in herbalist shops in many countries (Phillips, 1992). NS seeds were reported to have antioxidant, antimalarial, antihyperglycemic, radioprotective, and immunomodulator properties (Assayed, 2010; Benhaddou-Andaloussi et al., 2008; Boskabady et al., 2011; Okeola et al., 2011). The neuronprotective effect of NS was demonstrated in experimental animal models (Kanter et al., 2006). The aim of this study was to investigate the possible harmful effects of FA on humoral immunity. In addition, the possible protective properties of NS oil against these adverse effects were investigated.
Materials and methods Animals and treatments
lighting (07.00–19.00 h). All the rats were allowed to acclimatize for a week before the experimentation. The rats were divided into five groups. Clean air was provided in an FA-free environment for 4 weeks to the rats in the control group (n ¼ 5). The other 28 rats were divided into four study groups. Group FA1 was exposed to FA (5 ppm/8 h/day) for 4 weeks (n ¼ 7), group FA þ NS1 was treated with NS oil (1 ml/kg/day by gavage) and exposed to FA (5 ppm) inhalation for 4 weeks (n ¼ 7), group FA2 was exposed to FA (10 ppm/8 h/day), and group FA þ NS2 was treated with NS oil (1 ml/kg/day by gavage) and exposed to FA (10 ppm) inhalation for 4 weeks (n ¼ 7). The concentrations of FA were those described by Ozen et al. (2005), and the FA exposure periods were those described by Ozen et al. (2002) and Sarsilmaz et al. (2007). The dose of NS was based on a previous study (Assayed, 2010). The rats inhaled FA 5 days per week at regular intervals in glass cages through two holes that were made in each cage to allow the entry and exit of air. During non-FA exposure periods, the rats were taken from their glass cages and returned to their own cages where they received standard laboratory chow (supplied from Elazig Feed Plant, Elazig, Turkey) and tap water ad libitum.
Production of FA and dose measurement Paraformaldehyde (Merck KGaA, 64271 Darmstadt, Germany) was heated at a temperature of 35–40 C and depolymerized (Chang et al., 1981) to obtain FA gas. Gaseous FA was pumped into the glass cages with the help of a pumping system, according to the exposure period and FA concentration. The FA concentration in the glass cages was continually monitored with an FA monitor (catalog no: ZDL-300, Environmental Sensors Co., Boca Raton, Florida, USA), as recommended by the Occupational Safety and Health Administration.
Sample collection and immunological study
Adult male Sprague Dawley rats (weighing 270–300 g, n ¼ 33) were obtained from the Experimental Research Unit, Firat University, Elazig, Turkey. The Local Ethics Committee for Animal Research approved the experimental protocol for this study, and all animals received humane care in compliance with the European Convention on Animal Care. The rats were housed in cages in an air-conditioned room with automatically regulated temperature (22 + 1 C) and
At the end of the study period, all the animals were killed by decapitation, and blood samples were collected in routine biochemical test tubes. The serum was separated by centrifugation and stored at 80 C until the total Igs and C3 titer levels were measured. Micro-enzyme-linked immunosorbent assay (ELISA) kits from the Kamiya Biomedical Company (Seattle, Washington, USA), plus a double antibody sandwich ELISA protocol and ELISA reader (ELx800 BioTek, Winooski, Vermont, USA), were
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Table 1. The levels of total IgA, IgM, IgG, and C3 (mean + SEM). Parameters
Control (n ¼ 5)
FA1 (n ¼ 7)
FA2 (n ¼ 7)
FA þ NS1 (n ¼ 7)
FA þ NS2 (n ¼ 7)
IgA (ng/ml)
59,411.58 + 3769.05 10,572.10 + 401.76 72,680.16 + 13715.65 16,421.98 + 610.53
107,754.10 + 9768.07a 17,685.13 + 849.52a 33,186.97 + 9454.43a 18,267.14 + 292.20a
109,556.23 + 8452.60a 20,486.19 + 1045.85a 32,424.00 + 3205.56a 19,198.73 + 1094.12a
61,873.96 + 5,081.54b 10,743.76 + 595.21b 37,124.31 + 4915.13a 16,591.29 + 141.23b
73,354.39 + 1985.21a,c 10,458.51 + 1160.19c 29,415.94 + 2973.03a 17,292.86 + 153.42
IgM (ng/ml) IgG (ng/ml) C3 (ng/ml)
IgA: immunoglobulin A; IgM: immunoglobulin M; IgG: immunoglobulin G; C3: complement 3; FA: formaldehyde; NS: Nigella sativa. a p < 0.05: compared with control. b p < 0.05: compared with FA1. c p < 0.05: compared with FA2.
used to determine the titer levels of total IgA, IgM, IgG, and C3 in the serum.
Statistical analysis A Kruskal–Wallis test was used because the data did not show a normal distribution. For multiple comparisons, a Mann–Whitney U test was used. Quantitative data were expressed as the mean + standard error of the mean. The level of significance was set at p < 0.05.
Results Total IgA and IgM levels increased in the FA-exposed groups (dose dependent) when compared with the control group (p < 0.05). Total IgA levels also increased significantly in the FA þ NS2 group compared with the control group (p < 0.05). When the FA groups were compared with the FA þ NS groups, there was a significant decrease in total IgA and IgM levels in the FA þ NS1 group compared with those in the FA1 group (p < 0.05). Similarly, these Igs were decreased in the FA þ NS2 group compared with those in the FA2 group (p < 0.05; Table 1; Figures 1 and 2). Based on total IgG levels, the secondary immune response decreased in all the FA inhalation groups when compared with the control group (p < 0.05). The administration of NS did not change the IgG levels in the FA þ NS1 and FA þ NS2 groups compared with the FA groups (Table 1; Figure 3). Comparison of the C3 levels of the groups exposed only to FA with those of the control group revealed a significant increase in the former (p < 0.05). Furthermore, the C3 levels of the FA þ NS1 group decreased compared with those of the FA1 group (p < 0.05).
Figure 1. The levels of the serum total IgA of all groups. a p < 0.05: compared with control; bp < 0.05: compared with FA1; and cp < 0.05: compared with FA2. IgA: immunoglobulin A; FA: formaldehyde; NS: Nigella sativa.
Figure 2. The levels of the serum total IgM of all groups. a p < 0.05: compared with control; bp < 0.05: compared with FA1; and cp < 0.05: compared with FA2. IgM: immunoglobulin M; FA: formaldehyde; NS: Nigella sativa.
However, there were no significant differences in C3 levels between the FA2 and FA þ NS2 groups (Table 1; Figure 4).
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Figure 3. The levels of the serum total IgG of all groups. a p < 0.05: compared with control. IgG: immunoglobulin G; NS: Nigella sativa.
Figure 4. The levels of the serum C3 of all groups. ap < 0.05: compared with control and bp < 0.05: compared with FA1. C3: complement 3; FA: formaldehyde; NS: Nigella sativa.
Discussion There are many experimental studies on the effects of different chemical agents on the immune system (Ayatollahi M, 2002; Bogadi-Sare et al., 2000; Lange et al., 1973; Mishra et al., 2006; Zhu et al., 2011). However, only a limited number of studies have investigated the effect of FA on the immune system (Baj et al., 1994; Dean et al., 1984; Kuo et al., 1997; Veraldi et al., 2006). One study reported that immune and hematopoietic systems may be affected if they are exposed to FA for a long time (Baj et al., 1994). However, another reported that bone marrow cellularity, lymphoid organ weights, and hematological parameters were unchanged in mice exposed to 15 ppm of FA for 21 days (Dean et al., 1984). Another study found that although FA binds to and is transported by albumin, the immune system is capable of producing an antibody against this complex (Coutinho et al., 1995). Several studies showed that serum IgA levels increased following exposure to various toxic agents
(Mishra et al., 2006; Zhu et al., 2011). However, some researchers reported that occupational exposure to certain toxic agents decreased serum IgA levels (BogadiSare et al., 2000; Lange et al., 1973; Oh et al., 2005). Mishra et al. (2006) found that serum IgA levels were increased in a group of lead-exposed workers compared to those of unexposed healthy controls. In an experimental study, levels of IgA increased in rats exposed to aluminum (Zhu et al., 2011). Several studies showed that IgA levels decreased in workers with occupational exposure to benzene or dioxin (BogadiSare et al., 2000; Lange et al., 1973; Oh et al., 2005). In our study, serum IgA levels increased significantly in response to FA inhalation. IgA is a mucosaprotecting antibody. Based on the current findings, we conclude that IgA levels increase in response to FA exposure. Terro´n et al. (2009) showed that IgA levels decreased in response to antioxidant administration. In this study, the administration of NS oil decreased total serum IgA levels, which increased following FA exposure. NS ameliorated the immunotoxic effects of low-dose FA, but NS did not ameliorate the immunotoxic effects of the high-dose FA exposure. In an experimental study, rats that received FA orally (20, 40, and 80 mg/kg) showed a dosedependent decrease in serum IgM levels (Vargova´ et al., 1993). Serum IgM levels decreased in response to exposure to some toxic agents (Oh et al., 2005; Zhu et al., 2011), whereas serum IgM levels increased in response to exposure to some other toxic agents (Bogadi-Sare et al., 2000; Lange et al., 1973). In our study, the IgM values of the FA groups were elevated compared with those of the control group. We assume that the increase in IgM values in this study may have been an acute response of the immune system to the exposure to FA. In an experimental study, the serum IgM levels of birds treated with melatonin increased (Terro´n et al., 2009). In our study, the administration of NS oil decreased the serum total IgM levels, which were elevated in response to FA exposure. The acute immune response (IgM) caused both by the low- and high-dose FA returned to normal levels following the NS treatment. Aluminum exposure has been shown to increase serum IgG levels in rats (Zhu et al., 2011). However, oral administration of FA decreased serum IgG levels in rats in a dose-dependent manner (Vargova´ et al., 1993). Several studies of benzene and dioxin reported a dose-dependent decrease in serum IgG values (Lange et al., 1973; Oh et al., 2005). Ayatollahi (2002) found a significant reduction in the IgG levels of humans with
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occupational exposure to lead. Similarly, we found a significant decline in the IgG levels of the rats exposed to FA compared with those of the control group. This result suggests that FA inhalation substantially suppressed the secondary immune antibody response (IgG). In another study, melatonin administration increased serum levels of IgG compared to their control values in birds (Terro´n et al., 2009). In accordance with our findings, the NS oil did not improve the serum IgG levels, which were depressed by FA exposure. Several studies showed that dioxin exposure can increase C levels (Mocarelli et al., 1986; Ott et al., 1994). On the other hand, rats exposed to aluminum had decreased levels of C3 and C4 (Zhu et al., 2011). We found a significant increase in the serum C3 levels of the FA groups compared with the control group. This suggests that there is an important humoral immune response to FA. The return of elevated C3 levels following FA exposure to normal values after the administration of the NS oil suggests that cells that produce C3 are not irrevocably damaged by FA (reversible effect). In another study of rats challenged with typhoid antigen, treatment with NS volatile oil reduced the serum antibody titer compared with the control animals (Islam et al., 2004). Likewise, in this study, FA exposure increased serum IgA and IgM levels. These antibodies were decreased in the FA þ NS group compared with those of the FA groups.
Conclusion The acute antibody response (IgA and IgM) and C3 levels in serum increased significantly in the FA groups (dose dependent) compared with the control group. Moreover, the secondary immune response (IgG) of the FA groups decreased compared with the control group. The elevated responses of C and acute antibody to FA, which is a foreign antigen for the organism, returned to normal levels following the NS treatment (immunoregulatory effect). However, NS had no effect on the secondary immune response. Long-term studies with larger numbers of animals and with doses closer to those observed in humans are required to determine the immunotoxic effects of FA, the protective effects of NS, and related cellular and humoral immune parameters. Authors’ Note This study was presented partially at Joint Meeting of Anatomical Societies, 19–22 May 2011, Bursa, Turkey, as a poster.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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