http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–4 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.905590
RESEARCH ARTICLE
Oral isotretinoin therapy of acne patients decreases serum paraoxonase-1 activity through increasing oxidative stress Hatice Uce Ozkol1, Halil Ozkol2, Ayse Serap Karadag3, Serap Gunes Bilgili1, Yasin Tuluce2, and Omer Calka1 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
1
Faculty of Medicine, Department of Dermatology, Yuzuncu Yil University, Van, Turkey, 2Faculty of Medicine, Department of Medical Biology, Yuzuncu Yıl University, Van, Turkey, and 3Faculty of Medicine, Department of Dermotology, Medeniyet University, _Istanbul, Turkey
Abstract
Keywords
Objectives: There are only a few earlier studies suggesting relationship between isotretinoin treatment and oxidative stress however, their results are conflicting. Therefore we aimed to concretize the influence of isotretinoin treatment on oxidant/antioxidant status together with paraoxonase-1 (PON1) activity for the first time. Methods: The study was performed on serum samples obtained from 35 acne vulgaris patients before and after three months of isotretinoin treatment. PON1 activity, total oxidant status (TOS), total antioxidant capacity (TAC), oxidative stress index (OSI) and some routine biochemical parameters were monitored. Results: Dramatically decreased PON1 activity (p50.001), increased TOS level and OSI value (p50.001 and p50.001; respectively) as well as slightly diminished TAC level were noted in posttreatment stage. Moreover significant increases were observed in lactate dehydrogenase and gamma glutamyl transpeptidase activities and levels of total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, triglycerides, low density lipoprotein cholesterol/high density lipoprotein cholesterol ratio respectively (p50.05, p50.001, p50.001, p50.001, p50.001, p50.001 and p50.001) while marked decrease was seen in high density lipoprotein cholesterol (p50.01). Conclusion: This study revealed that decreased PON1 activity and increased oxidative stress may have a crucial role in the pathogenesis of isotretinoin’s side effects. Further studies on a large number of patients are needed to verify these results.
Acne vulgaris, isotretinoin, lipid profile, oxidative stress, paraoxonase-1
Introduction Isotretinoin is a derivative of vitamin A. Since receiving US FDA approval in 1982, it has become the most widely used drug in the treatment and remission of persistent severe acne. Healing of 80–90% is provided after 4–5 months of treatment. Among many side effects of isotretinoin, the most commonly seen alterations are mucocutaneous ones. Other reported side effects are headache, changes in liver functions, escalation of triglyceride and cholesterol levels, arthralgias, myalgias, and bone toxicity (Brito et al., 2010; Goodfield et al., 2010). Some earlier studies have shown that vitamin A intake induces cellular oxidative stress (de Oliveira et al., 2007; Pasquali et al., 2009a, b, 2010). The hypothesis of present study is that since isotretinoin is a metabolite of vitamin A, it might affect oxidant/antioxidant status in a same way like vitamin A, and therefore oxidative stress may take part in pathogenesis of isotretinoin’s side effects. However, the
History Received 6 August 2013 Revised 22 February 2014 Accepted 13 March 2014 Published online 2 April 2014
results of some recent studies in this regard are conflicting. For instance, while total antioxidant capacity (TAC) which is accepted as an important biomarker of antioxidant system increased in study of Georgala et al. (2005) in which 45 days of oral isotretinoin treatment with 0.5 mg/kg daily intake was realized, unchanged level of TAC was informed by Yorulmaz Demir et al. (2012) where the same dose and treatment period was applied. Another study which was also conducted on human blood like foregoing two studies suggested that levels of some antioxidants including reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) increased significantly in parallel with lipid peroxidation after isotretinoin therapy (Erturan et al., 2012). The aim of our current study was to contribute to the elimination of aforementioned contradiction between isotretinoin treatment and oxidant/antioxidant status and evaluate the activity of antioxidant enzyme paraoxonase-1 (PON1) for the first time in the literature in patients with acne vulgaris.
Materials and methods Address for correspondence: Hatice Uce Ozkol, MD, Yuzuncu Yil University, Faculty of Medicine, Department of Dermatology, 65100, Van, Turkey Tel: 0 90 432 215 76 07. Fax: 0 90 432 216 75 19. E-mail: [email protected], [email protected]
Subject The study was conducted in Department of Dermatology in Van Regional Research and Training Hospital and
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
2
H. U. Ozkol et al.
Department of Medical Biology in Medical Faculty of Yuzuncu Yil University between October 2010 and April 2011. Thirty-five patients with acne vulgaris were enrolled in the research (8 men and 27 women; mean ± SD age 21.33 ± 3.36 years, range 18–30). In order to attenuate the side effects of isotretinoin the study was conducted on autumn and winter sessions. A study group was formed from male or non-pregnant female patients with persistent severe acne who had therapeutic failure with topical treatments and tetracyclines. Isotretinoin therapy was initiated at a daily dose of 0.5– 0.75 mg/kg body weight and continued at least five months. Monitoring for PON1 activity, oxidative stress biomarkers including TAC, total oxidant status (TOS) and oxidative stress index (OSI) as well as some routine biochemical constituents were realized just before initiation (pretreatment) and three months after treatment of isotretinoin (posttreatment). The study protocol was carried out in accordance with the Helsinki Declaration as revised in 2000. This protocol was approved by the Yuzuncu Yil University ethics committee. A written consent to participate in the study was obtained from the patients after they were thoroughly informed about the research details. Blood samples Analyses of all parameters monitored in present study were performed just before initiation and three months after treatment of isotretinoin. Following an overnight fast, blood samples were taken into empty tubes, and immediately kept on ice at 4 C. Afterwards the serum was separated from the cells through centrifugation at 3000 rpm for 15 minutes, and the routine biochemical parameters including fasting blood glucose, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT; EC 2.6.1.2), aspartate aminotransferase (AST; EC 2.6.1.1), lactate dehydrogenase (LDH; EC 1.1.1.27), gamma glutamyl transpeptidase (GGT; EC 2.3.2.2), creatine kinase (CK; EC 2.7.3.2), albumin, total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), very low density lipoprotein cholesterol (VLDL) and LDL/HDL ratio were measured immediately. The remaining serum samples were stored at 80 C until analysis of the PON1 (EC 3.1.8.1) activity and levels of TAC and TOS. Measurement of TAC level The serum TAC level were measured spectrophotometrically (Genesys 10 UV Scanning UV/VIS Spectrophotometer) at 660 nm using kits developed by Erel (2004). The method is depended on the bleaching of distinct color of a more constant 2,20 -azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] radical cation via antioxidants. The precision of this assay is lower than 3% and there is no interference from bilirubin, hemoglobin, citrate or EDTA. The results were given as mmol Trolox Equivalent/L. Measurement of TOS level The serum TOS level were assessed spectrophotometrically (Genesys 10 UV Scanning UV/VIS Spectrophotometer) at
Drug Chem Toxicol, Early Online: 1–4
530 nm using kits developed by Erel (2005). In this new colorimetric method, oxidants existing in the sample oxidized the ferrous ion-o-dianisidine complex into ferric ion. The reaction of oxidation was elevated by glycerol molecules profusely present in the reaction medium. The ferric ion and xylenol orange generated a colored complex. The results were given as micromolar hydrogen peroxide equivalent per liter (mmol H2O2 Equivalent/L). Calculation of OSI value The OSI was assessed according to study of Bolukbas et al. (2005). It was defined as the ratio of the TOS level to the TAC level. Specifically, OSI (arbitrary unit) ¼ TOS (mmol H2O2 Equiv./L)/TAC (mmol Trolox Equiv./L). Measurement of PON1 activity The serum activity of PON1 was assessed via using paraoxon as a substrate and measured spectrophotometrically (Genesys 10 UV Scanning UV/VIS Spectrophotometer) by escalations in the absorbance at 412 nm owing to the formation of 4-nitrophenol as already reported (Verit et al., 2008). The activity was assessed at 25 C by appending 50 mL serum into 1 mL Tris-HCl buffer (100 mM at pH 8.0) including 5.5 mM of paraoxon and 2 mM CaCl2. The rate of production of 4-nitrophenol was assigned at 412 nm. Activity of enzyme was assessed via using the molar extinction coefficient 17.100 M1 cm1. Measurement of routine biochemical parameters Fasting blood glucose, BUN, creatinine, ALT, AST, LDH, GGT, CK, albumin, total cholesterol, triglycerides, LDL, HDL, VLDL and LDL/HDL ratio were assessed by an automatic analyzer (COBAS INTEGRA 400 plus, Roche, Basel, Switzerland) using the kits. Statistical analysis Descriptive statistics are presented as Mean ± Standard deviation for studied characteristics. Paired t test was performed to compare initial (just before initiation) and final (after 3 months of isotretinoin treatment) values of the characteristics. Statistical significant level was considered to 5% and SPSS 13.0 statistical software (SPSS Inc., Chicago, IL) was used for all statistical computations.
Results After isotretinoin treatment of three months all parameters were re-evaluated and compared to their initial levels. Results for serum PON1 activity and oxidant/antioxidant status were shown in Table 1. Isotretinoin treatment led to remarkable decrement of PON1 activity (p50.001) and increment of TOS and OSI levels (p50.001). Furthermore slight decline of TAC values was observed after treatment (p40.05). Routine biochemical parameters of patients were given in Table 2. While isotretinoin therapy significantly increased activities of some serum enzymes such as LDH, GGT (p50.05 and p50.001; respectively), slightly increased the others including ALT, AST and CK (p40.05). With regard to the lipid profile, total cholesterol, triglycerides, LDL, VLDL
Isotretinoin therapy decreases serum PON1 activity
DOI: 10.3109/01480545.2014.905590
Table 1. Comparison of pretreatment and posttreatment PON1 activity and oxidant/antioxidant status (Mean ± SD). Parameters
Pretreatment
Posttreatment
p Value
PON1 (U/L) 267.13 ± 20.12 244.82 ± 27.17 50.001 TAC (mmolTrolox Equiv./L) 2.26 ± 0.70 2.12 ± 0.66 0.404 TOS (mmol H2O2 Equiv./L) 15.79 ± 2.91 18.50 ± 3.45 50.001 OSI (Arbitrary units) 7.61 ± 2.44 9.24 ± 2.04 50.001 Paired t test was performed to compare initial (just before initiation) and final (after 3 months of isotretinoin treatment) values of the characteristics. PON1, paraoxonase-1; TAC, total antioxidant capacity; TOS, total oxidant status; OSI, oxidative stress index.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
Table 2. Comparison of pretreatment and posttreatment routine biochemical characteristics (Mean ± SD). Parameters
Pretreatment
Posttreatment
p Value
Fasting blood glucose (mg/dL) BUN (mg/dL) Creatinine (mg/dL) ALT (U/L) AST (U/L) LDH (U/L) GGT (U/L) CK (U/L) Albumin (g/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) LDL (mg/dL) HDL (mg/dL) VLDL (mg/dL) LDL/HDL ratio
87.95 ± 8.51
90.69 ± 9.85
0.076
22.41 ± 7.47 0.66 ± 0.13 17.48 ± 12.50 21.19 ± 8.57 367.80 ± 41.59 12.29 ± 5.36 95.60 ± 56.90 4.73 ± 0.33 158.70 ± 28.68
20.80 ± 5.90 0.65 ± 0.12 18.35 ± 13.04 23.48 ± 5.68 387.97 ± 45.15 15.83 ± 6.84 94.61 ± 32.53 5.45 ± 3.76 179.30 ± 34.60
0.128 0.396 0.663 0.121 0.046 50.001 0.888 0.282 50.001
89.23 ± 31.11 84.43 ± 24.10 53.23 ± 10.47 17.94 ± 6.18 1.58 ± 0.6
121.54 ± 54.99 100.53 ± 30.95 49.28 ± 10.90 24.82 ± 10.83 2.03 ± 0.83
50.001 50.001 50.01 50.001 50.001
Paired t test was performed to compare initial (just before initiation) and final (after 3 months of isotretinoin treatment) values of the characteristics. BUN, blood ure nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyltransferase; CK, creatine kinase; LDH, lactate dehidrogenase; HDL, high density lipoprotein cholesterol; LDL, low density lipoprotein cholesterol; VLDL, very low density lipoprotein cholesterol; LDL/HDL ratio, low density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio.
and LDL/HDL ratio showed a similar trend of dramatically higher level in posttreatment period (p50.001) compared to initial values whereas HDL markedly diminished in this period (p50.01). There were no significant alterations in values of fasting blood glucose, renal function markers BUN and creatinine as well as serum protein albumin (p40.05) between pretreatment and posttreatment periods.
Discussion Ductal hypercornification, increased sebum production, inflammation and Propionibacterium acnes colonization are known to be the key factors in pathogenesis of acne. Isotretinoin is the only drug that copes with all these factors. In spite of such beneficial actions, its numerous side effects are present. Therefore, efforts are needed to reveal mechanisms taking part in the pathogenesis of side effects of isotretinoin. Although some previous studies informed oxidative stress to be one of these mechanisms, we encountered conflicting levels of oxidants and antioxidants related to
3
isotretinoin treatment in the literature (Davis et al., 1990; Georgala et al., 2005; Yorulmaz Demir et al., 2012). Present study was conducted to contribute to the elimination of this contradiction and concretize whether isotretinoin treatment induce oxidative stress. For this purpose initial and final (after three months of isotretinoin therapy) levels of TAC and TOS were monitored to evaluate the status of oxidative stress. In addition, OSI value was defined as the ratio of the TOS level to the TAC level. In posttreatment stage, while significant increases were observed in TOS and OSI, slight decrement of TAC was noted (Table 1). These alterations indicate inducing effect of isotretinoin on oxidative stress and they are in agreement with results of Yorulmaz Demir et al. (2012) in which also increased neutrophil activation was reported. Activated neutrophils are known to produce reactive oxygen species (Akamatsu & Horio, 1998). In our study, dramatic increases of TOS level and OSI value may have been resulted from aforementioned activation. We encountered a few studies in the literature in which various biomarkers were evaluated to investigate influence of isotretinoin therapy on oxidative stress. But we did not see TOS and OSI among these biomarkers in any of foregoing studies while TAC was monitored in Georgala et al. (2005) and Yorulmaz Demir et al. (2012). However results of TAC obtained from these two studies are conflicting. Slightly diminished levels of TAC that was observed in our study is in line with Yorulmaz Demir et al. (2012) whereas contradict with Georgala et al. (2005) in which increment of TAC level was reported. Reason for such discrepancy is not fully understood at the moment. Because the same dose of isotretinoin were performed in both studies and ours. But small number of patients (totally 18; 10 women and 8 men) included in the study of Georgala et al. (2005) might have led to this paradox. Our current results concerning increased oxidative stress after isotretinoin therapy are also in consistent with some earlier studies in which different biological materials except serum were used. Erturan et al. (2012) investigated the effect of two months of isotretinoin therapy on erythrocyte and plasma. Significant increases in erythrocyte lipid peroxidation, GSH and GSH-Px levels as well as marked decreases of plasma vitamin E and lipid peroxidation were reported. In study of Pasquali et al. (2009a) the effect of both therapeutic (1000 and 2500 IU/kg) and high (4500 and 9000 IU/kg) doses of vitamin A (isotretinoin analog) on lungs of rats were monitored. Marked increment of lipid peroxidation and changed activities of antioxidant enzymes such as superoxide dismutase and catalase were measured. Another study which was realized by the same author revealed that treatment with low doses of vitamin A (100, 200 and 300 IU) throughout pregnancy and lactation caused an escalation of lipid peroxidation as well as catalase and superoxide dismutase activities in lungs of neonatal rats (Pasquali et al., 2010). Also, in some other previous studies enhanced lipid peroxidation levels and GSH-Px activity were informed after treatment with different doses of vitamin A (Besa et al., 1990; Dimery et al., 1997). PON1 is a powerful antioxidant enzyme however excessive oxidative stress reduces its activity (Mackness & Durrington, 1995; Mackness et al., 1993; Parthasarathy et al., 1990).
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
4
H. U. Ozkol et al.
PON1 enhances antiatherogenic effect of HDL and protects LDL from oxidation. Although dramatic changes of serum enzymes and lipid profile were reported after isotretinoin treatment in many previous studies (Brito et al., 2010), current study is the first trial evaluated foregoing parameters together with serum PON1 activity in acne vulgaris before and after three months of isotretinoin treatment. In our patient population, decreased serum PON1 activity may have been related to increased oxidative stress as evidenced from escalations of TOS level and OSI value after isotretinoin therapy (Table 1). As seen in Table 2, significant increases of serum total cholesterol, LDL, VLDL, LDL/HDL ratio and triglyceride levels as well as decreases of HDL are similar to results of some earlier studies (De Marchi et al., 2006; Erturan et al., 2012; Karadag et al., 2010; Zane et al., 2006). Present study also indicated a significant elevation in levels of some serum enzymes such as LDH and GGT similar to findings of Strauss et al. (2000). In conclusion, results of the current study concretized the formation of increased oxidative stress associated with isotretinoin treatment. Reduced PON1 activity may be related to such an imbalance in oxidant/antioxidant status. These alterations of oxidative stress and PON1 activity may have role in the pathogenesis of side effects of isotretinoin. Therefore, concomitant treatments of various antioxidants can be tested to alleviate such side effects. Long-term studies including a higher number of patients and cumulative isotretinoin doses are needed to approve our results.
Declaration of interest The authors have declared that there is no conflict of interest.
References Akamatsu H, Horio T. (1998). The possible role of reactive oxygen species generated by neutrophils in mediating acne inflammation. Dermatology 196:82–85. Besa EC, Abrahm JL, Bartholomew MJ, et al. (1990). Treatment with 13-cis-retinoic acid in transfusion-dependent patients with myelodysplastic syndrome and decreased toxicity with addition of alphatocopherol. Am J Med 89:739–747. Bolukbas C, Bolukbas FF, Horoz M, et al. (2005). Increased oxidative stress associated with the severity of the liver disease in various forms of hepatitis B virus infection. BMC Infect Dis 5:95. Brito Mde F, Sant’Anna IP, Galindo JC, et al. (2010). Evaluation of clinical adverse effects and laboratory alterations in patients with acne vulgaris treated with oral isotretinoin. An Bras Dermatol 85: 331–337. Davis WL, Crawford LA, Cooper OJ, et al. (1990). Generation of radical oxygen species by neural crest cells treated in vitro with isotretinoin and 4-oxo-isotretinoin. J Craniofac Genet Dev Biol 10:295–310. De Oliveira MR, Silvestrin RB, Mello EST, Moreira JC. (2007). Oxidative stress in the hip pocampus, anxiety-like behavior and
Drug Chem Toxicol, Early Online: 1–4
decreased locomotory and exploratory activity of adult rats: effects of subacute vitamin A supplementation at therapeutic doses. Neurotoxicology 28:1191–1199. De Marchi MA, Maranhao RC, Brandizzi LI, Souza DR. (2006). Effects of isotretinoin on the metabolism of triglyceride-rich lipoproteins and on the lipid profile in patients with acne. Arch Dermatol Res 297: 403–408. Dimery IW, Hong WK, Lee JJ, et al. (1997). Phase I trial of alphatocopherol effects on 13-cis-retinoic acid toxicity. Ann Oncol 8:85–89. Erel O. (2004). A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 37:277–285. Erel O. (2005). A new automated colorimetric method for measuring total oxidant status. Clin Biochem 38:1103–1111. _ Nazirog˘lu M, Akkaya VB. (2012). Isotretinoin treatment Erturan I, induces oxidative toxicity in blood of patients with acne vulgaris: a clinical pilot study. Cell Biochem Funct 30:552–557. Georgala S, Papassotiriou I, Georgala C, et al. (2005). Isotretinoin therapy induces DNA oxidative damage. Clin Chem Lab Med 43: 1178–1182. Goodfield MJ, Cox NH, Browser A, et al. (2010). Advice on the safe introduction and continued use of isotretinoin in acne in the U.K. 2010. Br J Dermatol 162:1172–1179. Karadag AS, Ertugrul DT, Tutal E, Akin KO. (2010). Short-term isotretinoin treatment decreases insulin-like growth factor-1 and insulin-like growth factor binding protein-3 levels: does isotretinoin affect growth hormone physiology? Br J Dermatol 162:798–802 Mackness MI, Durrington PN. (1995). High density lipoprotein, its enzymes and its potential to influence lipid peroxidation. Atherosclerosis 115:243–253. Mackness MI, Abbott C, Arrol S, Durrington PN. (1993). The role of high density lipoprotein and lipid-soluble antioxidant vitamins in inhibiting low-density lipoprotein oxidation. Biochem J 294:829–834. Parthasarathy S, Barnett J, Fong LG. (1990). High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein. Biochim Biophys Acta 1044:275–283. Pasquali MA, Gelain DP, de Oliveira MR, et al. (2009a). Vitamin A supplementation for different periods alters oxidative parameters in lungs of rats. J Med Food 12:1375–1380. Pasquali MA, Gelain DP, Oliveira MR, et al. (2009b). Vitamin A supplementation induces oxidative stress and decreases the immunocontent of catalase and superoxide dismutase in rat lungs. Exp Lung Res 35:427–438. Pasquali MA, Schnorr CE, Feistauer LB, et al. (2010). Vitamin A supplementation to pregnant and breastfeeding female rats induces oxidative stress in the neonatal lung. Reprod Toxicol 30:452–456. Strauss JS, Gottlieb AB, Jones T, et al. (2000). Concomitant administration of vitamin E does not change the side effects of isotretinoin as used in acne vulgaris: a randomized trial. J Am Acad Dermatol 43: 777–784. Verit FF, Erel O, Celik N. (2008). Serum paraoxonase-1 activity in women with endometriosis and its relationship with the stage of the disease. Hum Reprod 23:100–104. Yorulmaz Demir A, Metin A, Isıkoglu S, Erel O. (2012). The Effect of Isotretinoin on Oxidative Stress in Severe Acne Vulgaris Patients, Turkiye Klinikleri. J Med Sci 32:1026–1031. Zane LT, Leyden WA, Marqueling AL, Manos MM. (2006). A population based analysis of laboratory abnormalities during isotretinoin therapy for acne vulgaris. Arch Dermatol 142: 1016–1022.
RESEARCH ARTICLE
Oral isotretinoin therapy of acne patients decreases serum paraoxonase-1 activity through increasing oxidative stress Hatice Uce Ozkol1, Halil Ozkol2, Ayse Serap Karadag3, Serap Gunes Bilgili1, Yasin Tuluce2, and Omer Calka1 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
1
Faculty of Medicine, Department of Dermatology, Yuzuncu Yil University, Van, Turkey, 2Faculty of Medicine, Department of Medical Biology, Yuzuncu Yıl University, Van, Turkey, and 3Faculty of Medicine, Department of Dermotology, Medeniyet University, _Istanbul, Turkey
Abstract
Keywords
Objectives: There are only a few earlier studies suggesting relationship between isotretinoin treatment and oxidative stress however, their results are conflicting. Therefore we aimed to concretize the influence of isotretinoin treatment on oxidant/antioxidant status together with paraoxonase-1 (PON1) activity for the first time. Methods: The study was performed on serum samples obtained from 35 acne vulgaris patients before and after three months of isotretinoin treatment. PON1 activity, total oxidant status (TOS), total antioxidant capacity (TAC), oxidative stress index (OSI) and some routine biochemical parameters were monitored. Results: Dramatically decreased PON1 activity (p50.001), increased TOS level and OSI value (p50.001 and p50.001; respectively) as well as slightly diminished TAC level were noted in posttreatment stage. Moreover significant increases were observed in lactate dehydrogenase and gamma glutamyl transpeptidase activities and levels of total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, triglycerides, low density lipoprotein cholesterol/high density lipoprotein cholesterol ratio respectively (p50.05, p50.001, p50.001, p50.001, p50.001, p50.001 and p50.001) while marked decrease was seen in high density lipoprotein cholesterol (p50.01). Conclusion: This study revealed that decreased PON1 activity and increased oxidative stress may have a crucial role in the pathogenesis of isotretinoin’s side effects. Further studies on a large number of patients are needed to verify these results.
Acne vulgaris, isotretinoin, lipid profile, oxidative stress, paraoxonase-1
Introduction Isotretinoin is a derivative of vitamin A. Since receiving US FDA approval in 1982, it has become the most widely used drug in the treatment and remission of persistent severe acne. Healing of 80–90% is provided after 4–5 months of treatment. Among many side effects of isotretinoin, the most commonly seen alterations are mucocutaneous ones. Other reported side effects are headache, changes in liver functions, escalation of triglyceride and cholesterol levels, arthralgias, myalgias, and bone toxicity (Brito et al., 2010; Goodfield et al., 2010). Some earlier studies have shown that vitamin A intake induces cellular oxidative stress (de Oliveira et al., 2007; Pasquali et al., 2009a, b, 2010). The hypothesis of present study is that since isotretinoin is a metabolite of vitamin A, it might affect oxidant/antioxidant status in a same way like vitamin A, and therefore oxidative stress may take part in pathogenesis of isotretinoin’s side effects. However, the
History Received 6 August 2013 Revised 22 February 2014 Accepted 13 March 2014 Published online 2 April 2014
results of some recent studies in this regard are conflicting. For instance, while total antioxidant capacity (TAC) which is accepted as an important biomarker of antioxidant system increased in study of Georgala et al. (2005) in which 45 days of oral isotretinoin treatment with 0.5 mg/kg daily intake was realized, unchanged level of TAC was informed by Yorulmaz Demir et al. (2012) where the same dose and treatment period was applied. Another study which was also conducted on human blood like foregoing two studies suggested that levels of some antioxidants including reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) increased significantly in parallel with lipid peroxidation after isotretinoin therapy (Erturan et al., 2012). The aim of our current study was to contribute to the elimination of aforementioned contradiction between isotretinoin treatment and oxidant/antioxidant status and evaluate the activity of antioxidant enzyme paraoxonase-1 (PON1) for the first time in the literature in patients with acne vulgaris.
Materials and methods Address for correspondence: Hatice Uce Ozkol, MD, Yuzuncu Yil University, Faculty of Medicine, Department of Dermatology, 65100, Van, Turkey Tel: 0 90 432 215 76 07. Fax: 0 90 432 216 75 19. E-mail: [email protected], [email protected]
Subject The study was conducted in Department of Dermatology in Van Regional Research and Training Hospital and
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
2
H. U. Ozkol et al.
Department of Medical Biology in Medical Faculty of Yuzuncu Yil University between October 2010 and April 2011. Thirty-five patients with acne vulgaris were enrolled in the research (8 men and 27 women; mean ± SD age 21.33 ± 3.36 years, range 18–30). In order to attenuate the side effects of isotretinoin the study was conducted on autumn and winter sessions. A study group was formed from male or non-pregnant female patients with persistent severe acne who had therapeutic failure with topical treatments and tetracyclines. Isotretinoin therapy was initiated at a daily dose of 0.5– 0.75 mg/kg body weight and continued at least five months. Monitoring for PON1 activity, oxidative stress biomarkers including TAC, total oxidant status (TOS) and oxidative stress index (OSI) as well as some routine biochemical constituents were realized just before initiation (pretreatment) and three months after treatment of isotretinoin (posttreatment). The study protocol was carried out in accordance with the Helsinki Declaration as revised in 2000. This protocol was approved by the Yuzuncu Yil University ethics committee. A written consent to participate in the study was obtained from the patients after they were thoroughly informed about the research details. Blood samples Analyses of all parameters monitored in present study were performed just before initiation and three months after treatment of isotretinoin. Following an overnight fast, blood samples were taken into empty tubes, and immediately kept on ice at 4 C. Afterwards the serum was separated from the cells through centrifugation at 3000 rpm for 15 minutes, and the routine biochemical parameters including fasting blood glucose, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT; EC 2.6.1.2), aspartate aminotransferase (AST; EC 2.6.1.1), lactate dehydrogenase (LDH; EC 1.1.1.27), gamma glutamyl transpeptidase (GGT; EC 2.3.2.2), creatine kinase (CK; EC 2.7.3.2), albumin, total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), very low density lipoprotein cholesterol (VLDL) and LDL/HDL ratio were measured immediately. The remaining serum samples were stored at 80 C until analysis of the PON1 (EC 3.1.8.1) activity and levels of TAC and TOS. Measurement of TAC level The serum TAC level were measured spectrophotometrically (Genesys 10 UV Scanning UV/VIS Spectrophotometer) at 660 nm using kits developed by Erel (2004). The method is depended on the bleaching of distinct color of a more constant 2,20 -azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] radical cation via antioxidants. The precision of this assay is lower than 3% and there is no interference from bilirubin, hemoglobin, citrate or EDTA. The results were given as mmol Trolox Equivalent/L. Measurement of TOS level The serum TOS level were assessed spectrophotometrically (Genesys 10 UV Scanning UV/VIS Spectrophotometer) at
Drug Chem Toxicol, Early Online: 1–4
530 nm using kits developed by Erel (2005). In this new colorimetric method, oxidants existing in the sample oxidized the ferrous ion-o-dianisidine complex into ferric ion. The reaction of oxidation was elevated by glycerol molecules profusely present in the reaction medium. The ferric ion and xylenol orange generated a colored complex. The results were given as micromolar hydrogen peroxide equivalent per liter (mmol H2O2 Equivalent/L). Calculation of OSI value The OSI was assessed according to study of Bolukbas et al. (2005). It was defined as the ratio of the TOS level to the TAC level. Specifically, OSI (arbitrary unit) ¼ TOS (mmol H2O2 Equiv./L)/TAC (mmol Trolox Equiv./L). Measurement of PON1 activity The serum activity of PON1 was assessed via using paraoxon as a substrate and measured spectrophotometrically (Genesys 10 UV Scanning UV/VIS Spectrophotometer) by escalations in the absorbance at 412 nm owing to the formation of 4-nitrophenol as already reported (Verit et al., 2008). The activity was assessed at 25 C by appending 50 mL serum into 1 mL Tris-HCl buffer (100 mM at pH 8.0) including 5.5 mM of paraoxon and 2 mM CaCl2. The rate of production of 4-nitrophenol was assigned at 412 nm. Activity of enzyme was assessed via using the molar extinction coefficient 17.100 M1 cm1. Measurement of routine biochemical parameters Fasting blood glucose, BUN, creatinine, ALT, AST, LDH, GGT, CK, albumin, total cholesterol, triglycerides, LDL, HDL, VLDL and LDL/HDL ratio were assessed by an automatic analyzer (COBAS INTEGRA 400 plus, Roche, Basel, Switzerland) using the kits. Statistical analysis Descriptive statistics are presented as Mean ± Standard deviation for studied characteristics. Paired t test was performed to compare initial (just before initiation) and final (after 3 months of isotretinoin treatment) values of the characteristics. Statistical significant level was considered to 5% and SPSS 13.0 statistical software (SPSS Inc., Chicago, IL) was used for all statistical computations.
Results After isotretinoin treatment of three months all parameters were re-evaluated and compared to their initial levels. Results for serum PON1 activity and oxidant/antioxidant status were shown in Table 1. Isotretinoin treatment led to remarkable decrement of PON1 activity (p50.001) and increment of TOS and OSI levels (p50.001). Furthermore slight decline of TAC values was observed after treatment (p40.05). Routine biochemical parameters of patients were given in Table 2. While isotretinoin therapy significantly increased activities of some serum enzymes such as LDH, GGT (p50.05 and p50.001; respectively), slightly increased the others including ALT, AST and CK (p40.05). With regard to the lipid profile, total cholesterol, triglycerides, LDL, VLDL
Isotretinoin therapy decreases serum PON1 activity
DOI: 10.3109/01480545.2014.905590
Table 1. Comparison of pretreatment and posttreatment PON1 activity and oxidant/antioxidant status (Mean ± SD). Parameters
Pretreatment
Posttreatment
p Value
PON1 (U/L) 267.13 ± 20.12 244.82 ± 27.17 50.001 TAC (mmolTrolox Equiv./L) 2.26 ± 0.70 2.12 ± 0.66 0.404 TOS (mmol H2O2 Equiv./L) 15.79 ± 2.91 18.50 ± 3.45 50.001 OSI (Arbitrary units) 7.61 ± 2.44 9.24 ± 2.04 50.001 Paired t test was performed to compare initial (just before initiation) and final (after 3 months of isotretinoin treatment) values of the characteristics. PON1, paraoxonase-1; TAC, total antioxidant capacity; TOS, total oxidant status; OSI, oxidative stress index.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
Table 2. Comparison of pretreatment and posttreatment routine biochemical characteristics (Mean ± SD). Parameters
Pretreatment
Posttreatment
p Value
Fasting blood glucose (mg/dL) BUN (mg/dL) Creatinine (mg/dL) ALT (U/L) AST (U/L) LDH (U/L) GGT (U/L) CK (U/L) Albumin (g/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) LDL (mg/dL) HDL (mg/dL) VLDL (mg/dL) LDL/HDL ratio
87.95 ± 8.51
90.69 ± 9.85
0.076
22.41 ± 7.47 0.66 ± 0.13 17.48 ± 12.50 21.19 ± 8.57 367.80 ± 41.59 12.29 ± 5.36 95.60 ± 56.90 4.73 ± 0.33 158.70 ± 28.68
20.80 ± 5.90 0.65 ± 0.12 18.35 ± 13.04 23.48 ± 5.68 387.97 ± 45.15 15.83 ± 6.84 94.61 ± 32.53 5.45 ± 3.76 179.30 ± 34.60
0.128 0.396 0.663 0.121 0.046 50.001 0.888 0.282 50.001
89.23 ± 31.11 84.43 ± 24.10 53.23 ± 10.47 17.94 ± 6.18 1.58 ± 0.6
121.54 ± 54.99 100.53 ± 30.95 49.28 ± 10.90 24.82 ± 10.83 2.03 ± 0.83
50.001 50.001 50.01 50.001 50.001
Paired t test was performed to compare initial (just before initiation) and final (after 3 months of isotretinoin treatment) values of the characteristics. BUN, blood ure nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyltransferase; CK, creatine kinase; LDH, lactate dehidrogenase; HDL, high density lipoprotein cholesterol; LDL, low density lipoprotein cholesterol; VLDL, very low density lipoprotein cholesterol; LDL/HDL ratio, low density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio.
and LDL/HDL ratio showed a similar trend of dramatically higher level in posttreatment period (p50.001) compared to initial values whereas HDL markedly diminished in this period (p50.01). There were no significant alterations in values of fasting blood glucose, renal function markers BUN and creatinine as well as serum protein albumin (p40.05) between pretreatment and posttreatment periods.
Discussion Ductal hypercornification, increased sebum production, inflammation and Propionibacterium acnes colonization are known to be the key factors in pathogenesis of acne. Isotretinoin is the only drug that copes with all these factors. In spite of such beneficial actions, its numerous side effects are present. Therefore, efforts are needed to reveal mechanisms taking part in the pathogenesis of side effects of isotretinoin. Although some previous studies informed oxidative stress to be one of these mechanisms, we encountered conflicting levels of oxidants and antioxidants related to
3
isotretinoin treatment in the literature (Davis et al., 1990; Georgala et al., 2005; Yorulmaz Demir et al., 2012). Present study was conducted to contribute to the elimination of this contradiction and concretize whether isotretinoin treatment induce oxidative stress. For this purpose initial and final (after three months of isotretinoin therapy) levels of TAC and TOS were monitored to evaluate the status of oxidative stress. In addition, OSI value was defined as the ratio of the TOS level to the TAC level. In posttreatment stage, while significant increases were observed in TOS and OSI, slight decrement of TAC was noted (Table 1). These alterations indicate inducing effect of isotretinoin on oxidative stress and they are in agreement with results of Yorulmaz Demir et al. (2012) in which also increased neutrophil activation was reported. Activated neutrophils are known to produce reactive oxygen species (Akamatsu & Horio, 1998). In our study, dramatic increases of TOS level and OSI value may have been resulted from aforementioned activation. We encountered a few studies in the literature in which various biomarkers were evaluated to investigate influence of isotretinoin therapy on oxidative stress. But we did not see TOS and OSI among these biomarkers in any of foregoing studies while TAC was monitored in Georgala et al. (2005) and Yorulmaz Demir et al. (2012). However results of TAC obtained from these two studies are conflicting. Slightly diminished levels of TAC that was observed in our study is in line with Yorulmaz Demir et al. (2012) whereas contradict with Georgala et al. (2005) in which increment of TAC level was reported. Reason for such discrepancy is not fully understood at the moment. Because the same dose of isotretinoin were performed in both studies and ours. But small number of patients (totally 18; 10 women and 8 men) included in the study of Georgala et al. (2005) might have led to this paradox. Our current results concerning increased oxidative stress after isotretinoin therapy are also in consistent with some earlier studies in which different biological materials except serum were used. Erturan et al. (2012) investigated the effect of two months of isotretinoin therapy on erythrocyte and plasma. Significant increases in erythrocyte lipid peroxidation, GSH and GSH-Px levels as well as marked decreases of plasma vitamin E and lipid peroxidation were reported. In study of Pasquali et al. (2009a) the effect of both therapeutic (1000 and 2500 IU/kg) and high (4500 and 9000 IU/kg) doses of vitamin A (isotretinoin analog) on lungs of rats were monitored. Marked increment of lipid peroxidation and changed activities of antioxidant enzymes such as superoxide dismutase and catalase were measured. Another study which was realized by the same author revealed that treatment with low doses of vitamin A (100, 200 and 300 IU) throughout pregnancy and lactation caused an escalation of lipid peroxidation as well as catalase and superoxide dismutase activities in lungs of neonatal rats (Pasquali et al., 2010). Also, in some other previous studies enhanced lipid peroxidation levels and GSH-Px activity were informed after treatment with different doses of vitamin A (Besa et al., 1990; Dimery et al., 1997). PON1 is a powerful antioxidant enzyme however excessive oxidative stress reduces its activity (Mackness & Durrington, 1995; Mackness et al., 1993; Parthasarathy et al., 1990).
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Dalhousie University on 06/29/14 For personal use only.
4
H. U. Ozkol et al.
PON1 enhances antiatherogenic effect of HDL and protects LDL from oxidation. Although dramatic changes of serum enzymes and lipid profile were reported after isotretinoin treatment in many previous studies (Brito et al., 2010), current study is the first trial evaluated foregoing parameters together with serum PON1 activity in acne vulgaris before and after three months of isotretinoin treatment. In our patient population, decreased serum PON1 activity may have been related to increased oxidative stress as evidenced from escalations of TOS level and OSI value after isotretinoin therapy (Table 1). As seen in Table 2, significant increases of serum total cholesterol, LDL, VLDL, LDL/HDL ratio and triglyceride levels as well as decreases of HDL are similar to results of some earlier studies (De Marchi et al., 2006; Erturan et al., 2012; Karadag et al., 2010; Zane et al., 2006). Present study also indicated a significant elevation in levels of some serum enzymes such as LDH and GGT similar to findings of Strauss et al. (2000). In conclusion, results of the current study concretized the formation of increased oxidative stress associated with isotretinoin treatment. Reduced PON1 activity may be related to such an imbalance in oxidant/antioxidant status. These alterations of oxidative stress and PON1 activity may have role in the pathogenesis of side effects of isotretinoin. Therefore, concomitant treatments of various antioxidants can be tested to alleviate such side effects. Long-term studies including a higher number of patients and cumulative isotretinoin doses are needed to approve our results.
Declaration of interest The authors have declared that there is no conflict of interest.
References Akamatsu H, Horio T. (1998). The possible role of reactive oxygen species generated by neutrophils in mediating acne inflammation. Dermatology 196:82–85. Besa EC, Abrahm JL, Bartholomew MJ, et al. (1990). Treatment with 13-cis-retinoic acid in transfusion-dependent patients with myelodysplastic syndrome and decreased toxicity with addition of alphatocopherol. Am J Med 89:739–747. Bolukbas C, Bolukbas FF, Horoz M, et al. (2005). Increased oxidative stress associated with the severity of the liver disease in various forms of hepatitis B virus infection. BMC Infect Dis 5:95. Brito Mde F, Sant’Anna IP, Galindo JC, et al. (2010). Evaluation of clinical adverse effects and laboratory alterations in patients with acne vulgaris treated with oral isotretinoin. An Bras Dermatol 85: 331–337. Davis WL, Crawford LA, Cooper OJ, et al. (1990). Generation of radical oxygen species by neural crest cells treated in vitro with isotretinoin and 4-oxo-isotretinoin. J Craniofac Genet Dev Biol 10:295–310. De Oliveira MR, Silvestrin RB, Mello EST, Moreira JC. (2007). Oxidative stress in the hip pocampus, anxiety-like behavior and
Drug Chem Toxicol, Early Online: 1–4
decreased locomotory and exploratory activity of adult rats: effects of subacute vitamin A supplementation at therapeutic doses. Neurotoxicology 28:1191–1199. De Marchi MA, Maranhao RC, Brandizzi LI, Souza DR. (2006). Effects of isotretinoin on the metabolism of triglyceride-rich lipoproteins and on the lipid profile in patients with acne. Arch Dermatol Res 297: 403–408. Dimery IW, Hong WK, Lee JJ, et al. (1997). Phase I trial of alphatocopherol effects on 13-cis-retinoic acid toxicity. Ann Oncol 8:85–89. Erel O. (2004). A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 37:277–285. Erel O. (2005). A new automated colorimetric method for measuring total oxidant status. Clin Biochem 38:1103–1111. _ Nazirog˘lu M, Akkaya VB. (2012). Isotretinoin treatment Erturan I, induces oxidative toxicity in blood of patients with acne vulgaris: a clinical pilot study. Cell Biochem Funct 30:552–557. Georgala S, Papassotiriou I, Georgala C, et al. (2005). Isotretinoin therapy induces DNA oxidative damage. Clin Chem Lab Med 43: 1178–1182. Goodfield MJ, Cox NH, Browser A, et al. (2010). Advice on the safe introduction and continued use of isotretinoin in acne in the U.K. 2010. Br J Dermatol 162:1172–1179. Karadag AS, Ertugrul DT, Tutal E, Akin KO. (2010). Short-term isotretinoin treatment decreases insulin-like growth factor-1 and insulin-like growth factor binding protein-3 levels: does isotretinoin affect growth hormone physiology? Br J Dermatol 162:798–802 Mackness MI, Durrington PN. (1995). High density lipoprotein, its enzymes and its potential to influence lipid peroxidation. Atherosclerosis 115:243–253. Mackness MI, Abbott C, Arrol S, Durrington PN. (1993). The role of high density lipoprotein and lipid-soluble antioxidant vitamins in inhibiting low-density lipoprotein oxidation. Biochem J 294:829–834. Parthasarathy S, Barnett J, Fong LG. (1990). High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein. Biochim Biophys Acta 1044:275–283. Pasquali MA, Gelain DP, de Oliveira MR, et al. (2009a). Vitamin A supplementation for different periods alters oxidative parameters in lungs of rats. J Med Food 12:1375–1380. Pasquali MA, Gelain DP, Oliveira MR, et al. (2009b). Vitamin A supplementation induces oxidative stress and decreases the immunocontent of catalase and superoxide dismutase in rat lungs. Exp Lung Res 35:427–438. Pasquali MA, Schnorr CE, Feistauer LB, et al. (2010). Vitamin A supplementation to pregnant and breastfeeding female rats induces oxidative stress in the neonatal lung. Reprod Toxicol 30:452–456. Strauss JS, Gottlieb AB, Jones T, et al. (2000). Concomitant administration of vitamin E does not change the side effects of isotretinoin as used in acne vulgaris: a randomized trial. J Am Acad Dermatol 43: 777–784. Verit FF, Erel O, Celik N. (2008). Serum paraoxonase-1 activity in women with endometriosis and its relationship with the stage of the disease. Hum Reprod 23:100–104. Yorulmaz Demir A, Metin A, Isıkoglu S, Erel O. (2012). The Effect of Isotretinoin on Oxidative Stress in Severe Acne Vulgaris Patients, Turkiye Klinikleri. J Med Sci 32:1026–1031. Zane LT, Leyden WA, Marqueling AL, Manos MM. (2006). A population based analysis of laboratory abnormalities during isotretinoin therapy for acne vulgaris. Arch Dermatol 142: 1016–1022.
