The effect of maternal tobacco smoking and second-hand tobacco smoke exposure on human milk oxidant-antioxidant status

Author’s Accepted Manuscript The effect of maternal tobacco smoking and second-hand tobacco smoke exposure on human milk oxidant-antioxidant status Marta Napierala, Thurman Allen Merritt, Izabela Miechowicz, Katarzyna Mielnik, Jan Mazela, Ewa Florek www.elsevier.com/locate/envres

PII: DOI: Reference:

S0013-9351(18)30646-7 https://doi.org/10.1016/j.envres.2018.12.017 YENRS8198

To appear in: Environmental Research Received date: 4 July 2018 Revised date: 13 November 2018 Accepted date: 9 December 2018 Cite this article as: Marta Napierala, Thurman Allen Merritt, Izabela Miechowicz, Katarzyna Mielnik, Jan Mazela and Ewa Florek, The effect of maternal tobacco smoking and second-hand tobacco smoke exposure on human milk oxidant-antioxidant status, Environmental Research, https://doi.org/10.1016/j.envres.2018.12.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The effect of maternal tobacco smoking and second-hand tobacco smoke exposure on human milk oxidant-antioxidant status Tobacco smoke and milk oxidant-antioxidant status

Marta Napierala1, Thurman Allen Merritt2, Izabela Miechowicz3, Katarzyna Mielnik4, Jan Mazela4, Ewa Florek1* 1

Laboratory of Environmental Research, Department of Toxicology, Poznan University of Medical Sciences, 30 Dojazd Street, 60-631 Poznan, Poland 2 Children’s Hospital, School of Medicine, Loma Linda University, Loma Linda, California 92354, USA 3 Department of Computer Science and Statistics, Poznan University of Medical Sciences, 79 Dabrowskiego Street, 60-529 Poznan, Poland 4 Department of Newborns' Infectious Diseases, Poznan University of Medical Sciences, 33 Polna Street, 60-535 Poznan, Poland *Corresponding author: Prof. Ewa Florek, Ph.D., D.Sc., Head of Laboratory of Environmental Research, Department of Toxicology, University of Medical Sciences, 30 Dojazd Street, 60-631 Poznan, Poland; Phone (+48) 61 847 20 81, Fax (+48) 61 847 20 81 ext. 157, e-mail: [email protected]

ABSTRACT Background: Many women who smoke tobacco continue to do so during lactation, and many nonsmoking women are exposed to second-hand tobacco smoke (SHS) during the period that she wishes to breastfeed. There are reports documenting the adverse effects of maternal smoking during lactation on their infant's health; however, the pathophysiological mechanisms underlying these effects are incompletely understood. Objectives: Our study purpose was to examine the influence of tobacco smoke on biochemical markers reflecting the intensity of oxidative stress using concentration of total protein (TP), trolox equivalent antioxidant capacity (TEAC), S-nitrosothiols (RSNO), nitric oxide (NO), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), glutathione S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) in the plasma, colostrum, and mature milk of women who smoke, those only exposed to SHS, and non-smokers. Methods: Questionnaire data on the tobacco smoking status were verified based on the determination of cotinine by high performance liquid chromatography with diode array detector (HPLC-DAD). Relevant markers of oxidative stress and biochemical parameters were determined using spectrophotometric methods. 

The authors declare they have no actual or potential competing financial interests 1

Results: We found that tobacco smoking during lactation increases oxidative stress in the mother's plasma, colostrum, and mature milk, and lesser so in those exposed to SHS. Tobacco smoke significantly increase TBARS and decrease TEAC in colostrum and mature milk. In response to ROS generated by tobacco smoke increase the activity of antioxidant enzymes (SOD, GST, GPx and CAT), p<0.05. Discussion: Such exposure to tobacco smoke influences the antioxidant barrier of human colostrum and mature milk that can adversely affect their infant's health. Greater public health awareness of the adverse effects of tobacco smoking during lactation on breast milk quality and its protective effects is urgently needed. Graphical abstract

ABBREVIATIONS CAT, catalase; GPx, glutahione peroxidase; GSH, reduced gluthatione; GST, glutathione S– transferase; HPLC–DAD, high performance liquid chromatography with diode array detector; NO, nitric oxide; ONOO–, peroxynitrite; RNS, reactive nitrogen species; ROS, reactive oxygen species; RSNO, S–nitrosothiols; SHS, secondhand smoke; SIDS, sudden infant death syndrome; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TEAC, trolox equilvalent antioxidant capacity; TP, total proteins

Keywords: breast milk; lactation; tobacco smoking; second-hand tobacco smoke; oxidant-antioxidant status; oxidative stress 1. INTRODUCTION Many women who smoke tobacco continue to smoke during pregnancy and during breastfeeding. In the USA, 12–19% of women admit they smoke during pregnancy (Merritt et al., 2010; CDC, 2011a). It is estimated that in Europe this problem affects more than 1 in 10 pregnant women. Studies conducted in 29 European countries (>5.25 million pregnant women) show that smoking during pregnancy varied from under 5% in Lithuania and Sweden to 12.3% in Poland, 17.1% in France, and 19% in Scotland (Euro-Peristat, 2013). In most western countries, about 25% of women who smoke continue to do so during the third trimester of pregnancy. Worldwide approximately 50% of women who smoke do not stop using tobacco when they recognize that they are pregnant (Merritt et al., 2010). Furthermore, although many women will reduce or refrain from tobacco smoking while pregnant, after delivery of their baby up to 85% relapse back to smoking (Ashford, 2009; Napierała et al., 2

2016). Moreover, the prevalence of SHS exposure during pregnancy also appears to be high. The multi-country study in Latin America, Asia, and Africa (n = 7961) showed that SHS exposure is common among pregnant women and ranged from 17.1% in the Democratic Republic of the Congo to 91.6% in Pakistan (Bloch et al., 2008). Exact statics about exposure to tobacco smoke of women during lactation are not entirely known. Tobacco smoking during pregnancy increases the risk of fetal growth restriction, preterm delivery, preterm-related deaths and deaths from sudden infant death syndrome (SIDS) (CDC et al., 2013; Markunas et al., 2014; Pietryga et al., 2017; Ruisch et al., 2018). Moreover, early exposure to tobacco smoke can cause health outcomes during childhood and adolescence such as respiratory system diseases (Thacher et al., 2018), childhood overweight and obesity (Albers et al., 2018), diabetes (Fang et al., 2015), cancer (Rumrich et al., 2016), neurological and neurobehavioral disorders (Ekblad et al., 2015). There are reports regarding the adverse effect of smoking during lactation on the child's health, however the pathophysiological mechanisms underlying this are still not entirely known (Napierała et al., 2016). Breast milk provides all of the nutrients need by infants for healthy growth and development during the first 6 months after birth. Breastfeeding is associated with reduced risk of anemia, respiratory illnesses, otitis media, disease of the gastrointestinal tract during infancy, and secondary infections in infants born prematurely (Pecoraro et al., 2017). In additional to the nutritive properties of breast milk, it also provides natural antioxidant protection to the infant. These antioxidants and enzymes in human milk provide significant health benefits and exceed those of artificial milk in infant formulas marketed for feeding newborns and infants. However, there are controversies as to whether breast milk from women who continue to smoke, or use e-cigarettes during lactation or who are exposed to second-hand smoke during breastfeeding has similar benefits (Napierała et al., 2016). When tobacco is smoked reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated in the smoke and in the presence of water and physiologic fluids (serum, urine, formation of milk within the mammary glands). Excessive production of ROS and RNS result in oxidative stress and exceeds cellular capacity to detoxify these oxidants and repair cellular damage. The consequence of this imbalance results in the formation of peroxides and oxygenbased free radicals that damage DNA, proteins and lipids. Increased oxidative stress is now widely believed to be involved in the pathogenesis of many major diseases (Wooten et al., 2006). The effect of tobacco smoke (either active or second hand smoking) on the antioxidant properties of breast milk is still not well known. So far, there have been only few reports regarding this issue (Ortega et al., 1998a; Ortega et al., 1998b; Ermis et al., 2005; Zagierski et al., 2012; Shamsi et al., 2015). Our study objectives were to evaluate selected biochemical parameters reflecting the intensity of oxidative stress in plasma, colostrum, and mature milk in non-smoking mothers, mothers exposed to secondhand smoke during pregnancy and lactation, and women who smoke during the period of pregnancy and lactation after delivery of their baby. To our knowledge, there are no such comprehensive reports on the impact of tobacco smoke on human milk oxidant-antioxidant status.

2. METHODS

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2.1. Bioethics Committee approval The study protocol was approved by the Bioethics Committee under Act 593/13 of 13 June 2013, based on Polish legislation and Good Clinical Practice at the Poznan University of Medical Sciences. 2.2. Study groups The study included 150 healthy women aged 18–40, the patients of the Obstetrics and Gynecology Hospital at the Poznan University of Medical Sciences. Women participating in the studies were divided into 3 groups: non-smokers (control group); second-hand tobacco smokers; and actively smokers. The expected number of persons in each group, assumed 50 women. Questionnaire data on the tobacco smoking status were verified based on the determination of tobacco exposure biomarker (cotinine) in biological material (patients’ blood serum). Participation in the studies was voluntary and patients were informed about the aims and significance of the studies before they gave consent. 2.3. Survey questionnaire The first stage of the research was the author’s questionnaire regarding sociodemographic information and the women’s lifestyle. This study was carried out twice: first before sampling blood and colostrum (1 ± 2 days after delivery) and second time before sampling mature milk (30 ± 7 days after delivery). As a criterion for exclusion of the participating in the study, all parameters that could influence the concentration of the examined parameters were selected. Exclusion criteria were: chronic illness of mother, premature delivery, solid medications use, supplement use such as vitamins and minerals, use of illicit drugs, alcohol consumption, vegetarian or vegan diet, use of e-cigarettes or any form of nicotine replacement therapy. 2.4. Biological material Blood, colostrum and mature milk were sampled by voluntary consent of the mothers into sterile sample vials. Blood samples (8 mL) were taken from mothers one day after delivery as routine hematological tests and prepared specifically for biochemical (plasma) and toxicological tests (serum). The colostrum (3 mL) was sampled 1 ± 2 days after delivery and mature milk (3 mL) was sampled 30 ± 7 days after delivery. Material was divided and secured in sterile Eppendorf sample tubes. Milk samples intended for spectrophotometric measurements (except determining TBARS concentration) were defatted by centrifugation (2500 rpm for 20 minutes, at 4°C). All samples were stored at -80°C until the proper analyses began. Toxicological and biochemical determinations were performed in the Laboratory of Environmental Research at the Department of Toxicology from the Poznan University of Medical Sciences.

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2.5. Chemicals Certified Reference Materials (norephinedrine, cotinine) in the concentration of 1 mg/mL of methanol (Sigma Aldrich) were used to validate analytical measurement methods. Solvents used for chromatographic analysis were of HPLC grade (Sigma Aldrich). All chemicals used for biochemical determinations were of analytical reagent grade (Sigma Aldrich). 2.6. Extraction and determination of cotinine concentration Serum, colostrum, and mature milk (1 mL each) from each woman were used to determine tobacco smoke exposure marker (cotinine) concentration to verify the status of tobacco smoking indicated in the questionnaire. Cotinine content in the analyzed serum samples was determined by high performance liquid chromatography with diode array detector (HPLC-DAD). The internal standard (norephedrine) was prepared by diluting 1 mg/mL of certified in methanol solution to 100 µl/mL. Chromatography was preceded by extraction of the analyzed compound in a liquid–liquid system. The sodium hydroxide solution (0.2 mL of 0.1 M) was added to 1 mL of serum to obtain pH = 8, which was checked using a universal indicator. Then 150 µL of norephedrine in methanol solution at 100 µg/mL was added. Liquid–liquid type extraction from the prepared samples was performed. For that purpose, a mixture of dichloromethane and isopropanol at 9:1 (v/v) was added to the samples. Next, samples were shaken for 15 minutes in a Multi Bio RS24 shaker (Biosan). Then samples were centrifuged by 15 minutes at 4200 rpm (Centrifuge 5804/5804 R, Eppendorf). After centrifuging, 4 mL of the layer containing dichloromethane and isopropanol was sampled and transferred to a 7 mL borosilicate glass tube. The obtained extract, with 150 µL of hydrochloric acid 0.035 M in methanol, was evaporated under pressurized nitrogen. The dry residue was dissolved in 100 µL of mobile phase. Cotinine concentration in milk was determined by HPLC-DAD using procainamide as an internal standard. Chromatography was preceded by extraction of the analyzed compound in a liquid–liquid system. The internal standard was prepared by diluting 1 mg/mL procainamide in methanol solution to 100 µl/mL. The procainamide solution (10 µL of 100 µg/mL) and 1 mL of phosphate buffer (0.1 M) at pH = 6.8 were added to the milk sample. Liquid–liquid type extraction from the prepared samples was performed. For that purpose, 2.5 mL of chloroform and isopropanol mixture at 95:15 (v/v) was added to the samples. Next, samples were shaken for 10 minutes in a Multi Bio RS-24 shaker (Biosan). The next stage consisted in centrifuging for 10 minutes at 4200 rpm (Centrifuge 5804/5804 R, Eppendorf). After centrifuging, 2 mL of the layer containing chloroform–isopropanol was sampled and transferred to a 7 mL borosilicate glass tube. Extraction was repeated using the supernatant from the first extraction. Again, the layer containing the solvent mixture was sampled and transferred to the same tube. The obtained extract was evaporated until dry at 45°C. The dry residue was dissolved in 100 µL of mobile phase. Qualitative and quantitative analyses were performed using a liquid chromatograph by Agilent Technologies, series 1200 with diode array detector (Perlan Technologies). A liquid chromatography column with C8 silicone filling: 5 µm (particle size), 125 x 4 mm (length x ext. diameter) and precolumn: 5 µm, 4 x 4 mm (Lichrospher 60 RP-Select B by Merck) were used to separate the analyzed compounds. The conditions of chromatographic analysis are presented in Table 1.

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Table 1. Conditions of the HPLC analysis.

Parameter

Eluent

Cotinine determination in serum samples

Cotinine determination in milk samples

92%: mixture of 0.08 M sodium hydrogen phosphate buffer and 0,1 M potassium phosphate 0,5 mM triethylamine, pH = 4,2 buffer, pH = 5,0 (25ºC) 8%: acetonitrile

Flow rate

1 mL/min

1 mL/min

Pressure

≈ 1200 psi

≈ 1500 psi

30°C

30°C

Wavelength (λ)

260 nm

260 nm

Injection volume

20 µL

30 µL

Time of analysis

11 min

10 min

Temperature

The certified reference material used to optimize and validate the used methods was a calibration solution of cotinine in methanol at 1 mg/mL (Sigma-Aldrich). Standard curves were linear over the entire concentration range studied (10–1000 µg/mL). The methods used were characterized by a low limit of quantification (15 ng/mL, 12 ng/mL respectively for serum and milk), high recovery (ranging between 96 – 123% in serum and 96 – 109% in milk), and interday- (ranging between 2.10 – 7.34% in serum and 2.86 – 8.85% in milk) and intraday-repeatability (ranging between 3.76 – 8.41% in serum and 3.01 – 10.30% in milk). 2.7. Determination of oxidative stress markers and biochemical parameters Relevant markers of oxidative stress and biochemical parameters (TP, TEAC, RSNO, NO, TBARS, GSH, GST, GPx, SOD, CAT) were determined in plasma (1.5 mL), colostrum (1.5 mL), and mature milk (1.5 mL), using spectrophotometric methods. Total protein (TP) concentration was determined using Lowry’s method - a combination of a biuret test and Folin-Ciocalteu reaction (Lowry et al., 1951). The trolox equivalent antioxidant capacity (TEAC) of substances present in the solutions was measured based on the measurement of stable radical cation reduction capacity (ABTS)•+ (Konan et al., 2016). The concentration of S-nitrosothiols (RSNO) was determined using the Saville/Gries method (Saville et al., 1958, Wang et al., 2011). By measuring the concentration of stable degradation products, nitrates(V) and nitrates(III) (nitrites) in an aqueous solution, NO concentrations were

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determined (Bryan and Grisham, 2007). Thiobarbituric acid reactive substances (TBARS) measurement was used for monitoring lipid peroxidation (Rael et al., 2004). Quantitative determination of reduced glutathione (GSH) was performed using modified Ellman’s method with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) (Ellman, 1959). Glutathione S-transferase (GST) enzymatic activity was assessed based on the coupling reaction of thiol groups of L-glutathione with 1chloro-2,4-dinitrobenzene (CDBN) (Bartosz, 2013). The activity of glutathione peroxidase (GPx) was determined based on the oxidation reaction of glutathione in the presence of H2O2 to glutathione disulphide (GSSG), for which GPx is a catalyst. The assumed unit of GPx activity was the enzyme amount that oxidizes 1 µM of GSH (0.5 μM NADPH) within 1 minute (Flohé, 1984; Mannervik, 1985; Bartosz, 2013). The activity of superoxide dismutase (SOD) was determined using a method based on the capacity to inhibit adrenaline autoxidation by SOD. The assumed enzyme activity in the evaluated sample was the enzyme amount that causes 50% inhibition of adrenaline autoxidation to adrenochrome (A/min = 0.025) in the same analytic conditions (Bartosz, 2013). The activity of catalase (CAT) was determined based on the reaction of H 2O2 degradation. The unit of CAT activity is the enzyme amount that degrades 1 µM H2O2 solution within 1 minute, which corresponds to absorbance reduction by 0.036 U/min (volume: 1 mL, optical path length: 1 cm) (Bartosz, 2013). Three technical replicates were performed for each milk sample. 2.8. Statistical analysis of the results Test results were analyzed statistically using the Statistica 12 software by StatSoft and StatXact software by Cytel. Statistical significance was α = 0.05. A result was deemed statistically significant, if p < α. Variables measured on the interval scale (levels of oxidative stress parameters) are represented by mean, SD. Categorical variables were described by the number and%. For interval variables, their distributions were checked for normal distribution using the Lillieforse and Shapiro-Wilk tests. If the parameters were compared between the groups, in the case of noncompliance with the normal distribution, the Mann-Whitney or Kruskal-Wallis test was calculated. In the case of compliance with the normal distribution and equality of variance, the analysis of variance for unrelated samples or Student's t-test for unrelated samples were calculated. The Cochran-Cox test was used for variables with a distribution consistent with normal but not equal variance. Additionally, in order to determine between which groups there are differences, posthoc tests were used: Tuckey's test for variance analysis and Dunn's test for the Kruskal-Wallis test (Bender and Lange, 2001). To assess whether the level of parameters taken in different materials (plasma vs. colostrum, colostrum vs. specific milk) differ, the Student's t-test for related samples (in the case of compliance with the normal distribution) and the Wilcoxon test were calculated (in the case of non-compliance with the normal distribution ). In order to investigate the relationship between interval variables, due to noncompliance with the normal distribution, or for variables measured on the ordinal scale, the Spearman RS rank correlation coefficient was calculated. For variables with a normal distribution, the Pearson's linear correlation coefficient was calculated. 7

3. RESULTS 3.1. Declared smoking status and cotinine concentration Exposure to tobacco smoke was determined based on responses to the questionnaire data regarding the women’s smoking status, confirmed by determinations of main exposure biomarker (cotinine). The socio-economic characteristics of the studied women’s groups summarized in the Table 2. The statistically significant differences in the age and socio-economic status parameters distribution between the three analyzed groups were not observed. Table 2. Socioeconomic status of women in studied groups Non-smoking women (%) (n = 52)

Women exposed to SHS (%) (n = 51)

Tobacco smoking women (%) (n = 44)*

Age (years): 18-24 25-29 30-34 35-40

7.7 32.7 36.5 23.1

7.8 21.6 51.0 19.6

4.5 31.1 33.3 31.1

Education: Basic Vocational Medium Higher

0.0 7.7 25.0 67.3

7.2 10.4 23.7 58.7

10.1 16.4 20.6 52.9

Character of the work: Mental Physical Not working

76.9 15.5 7.6

65.6 31.2 3.2

54.1 30.3 15.6

2.0 26.9 71.1

6.3 18.1 75.6

10.2 31.3 58.5

61.6 28.8 9.6

52.2 32.6 15.2

55.5 28.9 15.6

Parameter

Economy status Low Medium High Number of children: 1 2 ≥3 * Some surveys were not complete

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The non-smoking group was comprised of women (n = 52) who declared to be nonsmokers before and during pregnancy and no cotinine was found in their biological material. In 8% (n = 12) of all women participating in the study and declaring non-smoking, cotinine was found in the sampled biological material. These patients were included in the group of smokers. Three women did not complete the questionnaire, and the analysis of biological material indicated that they were tobacco smokers. Among women qualified to the smoking group (n = 47), based on their declaration (Table 3) and/or the determined biomarker level (Figure 1), the observed cotinine concentration was: 38.39 ± 12.06 ng/mL of serum, 25.44 ± 7.98 ng/mL of colostrum (both collected at the same day after delivery), and 58.29 ± 23.09 ng/mL of mature milk (collected one month after delivery). Table 3. Summary of data collected using questionnaires regarding the tobacco smoking status and the intensity of smoking.

Parameter

Tobacco smoking women (%)

Smoking before pregnancy

100 (n = 32)

The amount of cigarettes smoked per day before pregnancy occasionally 1-5 6-10 11-16 >16

9,38 (n = 3) 21,88 (n = 7) 34,37 (n = 11) 18,75 (n = 6) 16,62 (n = 5)

Smoking during pregnancy Continuation Cessation

56,25 (n = 18) 43,75 (n = 16)

The amount of cigarettes smoked per day during pregnancy occasionally 1-5 6-10 11-16 >16

38,89 (n = 7) 22,22 (n = 4) 22,22 (n = 4) 11,11 (n = 2) 5,56 (n = 1)

Smoking during lactation (1 month after delivery) Continuation Cessation The amount of cigarettes smoked daily during lactation (1 month after delivery) occasionally 1-5 6-10 11-16

73,08 (n = 19) 26,92 (n = 7)

22,73 (n= 5) 22,73 (n = 5) 18,18 (n = 4) 22,73 (n = 5) 9

>16

13,63 (n = 3)

*They stopped smoking in the first or second trimester of pregnancy  Some women did not take part in the next stage of the research

Based on questionnaire, there was observed a tendency to rapid relapse to the addiction one month after delivery, in the case of women who stopped smoking during pregnancy. In the first month after delivery, about 16% of women started smoking again. In the group of women exposed to second-hand smoking (n = 51), mean cotinine concentration was 18.06 ± 2.60 ng/mL in serum, 13.32 ± 1.85 ng/mL in colostrum, and 16.61 ± 3.73 ng/mL in mature milk (mean ± SD).

Figure 1. Comparison of cotinine concentrations (ng/mL) in plasma *, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). a – statistically significant difference compared to the group of non-smoking women and exposed to SHS (p < 0.05)

The significant (p < 0.05), positive correlation between the cotinine concentration in serum and colostrum was observed both in group of smoking and second-hand smoking women (RS = 0.90, n = 28; RS = 0.84, n = 18; respectively). About 44% of the sampled population women admitted that their partners smoke tobacco (instead of their own smoking status) (Table 4). Table 4. Summary of data collected using questionnaires regarding the tobacco smoking status and the intensity of smoking by partners of women participating in the study.

Parameter

SHS exposure during pregnancy 

Nonsmoking women whose partners smoke (%)

Tobacco smoking women whose partners smoke (%)

86,27 (n = 44)

65,5 (n = 21)

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The amount of cigarettes smoked by partner occasionally 1-5 6-10 11-16 >16 SHS exposure during lactation (1 month after delivery)** The amount of cigarettes smoked by partner occasionally 1-5 6-10 11-16 >16

11,36 (n = 5) 15,91 (n = 7) 22,72 (n = 10) 20,45 (n = 9) 29,56 (n = 13)

14,29 (n= 3) 9,52 (n = 2) 14,29 (n = 3) 28,57 (n =6) 33,33 (n = 7)

82,05 (n = 32)

69,70 (n = 23)

12,50 (n= 4) 9,37 (n = 3) 31,25 (n = 10) 21,88 (n = 7) 25,00 (n = 8)

17,39 (n= 4) 17,39 (n = 4) 30,43 (n = 7) 13,05 (n = 3) 21,74 (n = 5)

Regarding only women whose partners smoked tobacco, not other cases of SHS exposure  Some women did not take part in the next stage of the research

3.2. Concentrations of oxidative stress markers and biochemical parameters Figures 2–11 present results for mean concentrations (with standard deviation, ±SD) of individual analyzed parameters in plasma and/or colostrum and mature milk among study groups, with all statistically significant differences. 3.2.1. Total protein The mean concentration of total protein for each tested material was comparable among the individual groups (Figure 2).

Figure 2. Comparison of TP concentrations (mg/mL) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). * 11

– statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.2.2. Trolox equivalent antioxidant capacity The total antioxidative potential was significantly lower in the serum and colostrum of SHS women (33.07 ± 10.86 and 88.26 ± 18.45 nM/mg, respectively) compared to non-smoking (44.11 ± 9.56 and 104.46 ± 26.12 nM/mg, respectively) (Figure 3). A significantly lower TAEC was observed in the mature milk of tobacco smoking women (105.24 ± 27.06 nM/mg) compared to SHS (120.05 ± 26.25 nM/mg). The level of TAEC in mature milk was significantly higher than in colostrum.

Figure 3. Comparison of TEAC concentrations (nM/mg of protein) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery)..a – statistically significant difference compared to the group of non-smoking women (p ≤ 0.0001). b – statistically significant difference compared to the group of women exposed to SHS (p ≤ 0.0001). * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.2.3. S-nitrosothiols The concentration of RSNO in plasma was statistically significantly higher in the group of tobacco smoking (13.04 ± 3.35 nM/mg) and SHS women (12.96 ± 3.64 nM/mg) compared to no-smoking (10.78 ± 2.47 nM/mg) (Figure 4). The concentration of RSNO in analyzed colostrum and mature milk samples was beyond the limit of the detection (0,1 - 2,5 mM).

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Figure 4. Comparison of RSNO concentrations (nM/mg of protein) in plasma of study groups. c – statistically significant difference compared to the group of non-smoking women (p = 0.0004)

3.2.4. Nitrogen oxide The concentration of NO in plasma (0.27 ± 0.09 nM/mg) and in colostrum (1.11 ± 0.66 nM/mg) of smoking women was significantly higher than in non-smoking group (0.21 ± 0.07 and 0.63 ± 0,32 nM/mg, respectively). Significant differences were also noted for plasma NO concentrations in the smoking group (0.27 ± 0.09 nM/mg), compared to SHS group (0.21 ± 0.10 nM/mg) and analogically in mature milk (2.27 ± 1.16 nM/mg vs. 1.53 ± 0.86 nM/mg, respectively) (Figure 5).

Figure 5. Comparison of NO concentrations (nM/mg of protein) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). b – statistically significant difference compared to the group of women exposed to SHS (p ≤ 0.0001). d – statistically significant difference compared to the group of non-smoking women (p = 0.0007). e – statistically significant difference compared to the group of women exposed to SHS (p = 0.0007). f – statistically significant difference compared to the group of non-smoking women (p = 0.0014). * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

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3.2.5. Thiobarbituric acid reactive substances In every tested material (plasma, colostrum and mature milk) the determined concentration of TBARS was significantly higher in the SHS group (0.11 ± 0.03; 0.33 ± 0.09; 0.38 ± 0.12 nM MDA/mg, respectively) than among women not exposed to tobacco smoke (0.09 ± 0.02; 0.26 ± 0.08; 0.32 ± 0.10 nM MDA/mg, respectively). Moreover, a higher TBARS level was demonstrated in plasma (0.13 ± 0.03 nM MDA/mg) and colostrum (0.41 ± 0.11 nM MDA/mg) of smoking women compared to non-smoking (0.09 ± 0.02; 0.26 ± 0.08 nM MDA/mg, respectively) and SHS women (0.11 ± 0.03; 0.33 ± 0.09 nM MDA/mg, respectively) (Figure 6).

Figure 6. Comparison of TBARS concentrations (nM MDA/mg of protein) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). a – statistically significant difference compared to the group of nonsmoking women (p ≤ 0.0001). b – statistically significant difference compared to the group of women exposed to SHS (p ≤ 0.0001). g – statistically significant difference compared to the group of nonsmoking women (p < 0.05). h – statistically significant difference compared to the group of women exposed to SHS (p < 0.05). * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.2.6. Reduced glutathione In the case of the determined GSH in plasma, no statistically significant differences were found. The obtained results were comparable in all study groups (3.78–3.98 nM/mg of protein) (Figure 7). The concentration of GSH in analyzed colostrum and mature milk samples was beyond the limit of the detection (0,09 - 2,25 mM).

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Figure 7. Comparison of GSH concentration (nM/mg of protein) in plasma of study groups. Statistically significant differences were not observed 3.2.7. Glutathione S-transferase The activity of GST both in colostrum (37.63 ± 5.91 nM/min/mg) and in mature milk (49.01 ± 9.79 nM/min/mg) of tobacco smoking women was significantly different than in the group of non-smoking women (34.03 ± 5.76 and 56.13 ± 10.59 nM/min/mg, respectively). No statistically significant difference were observed in plasma among individual study groups (Figure 8).

Figure 8. Comparison of GST concentration (nM/min/mg of protein) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). g – statistically significant difference compared to the group of non-smoking women (p < 0.05). i – statistically significant difference compared to the group of non-smoking women (p = 0.0142) . * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.2.8. Glutathione peroxidase A comparison of the determined GPx activity between the groups proved statistically significant differences among tobacco smoking women compared to the non15

smoking and second-hand smoking in all tested biological materials. The activity of GPx in plasma (3.04 ± 1.05 nM/min/mg) and in mature milk (4.29 ± 1.69 nM/min/mg) of smoking patient was significantly higher than that observed in non-smoking patients (2.37 ± 0.75 and 2.89 ± 1.14 nM/min/mg, respectively) and exposed to second hand tobacco smoke (2.49 ± 1.30 and 2.89 ± 0.95 nM/min/mg, respectively). The concentration of GPx in the colostrum of smoking women (1.68 ± 0.69 nM/min/mg) was significantly lower than in the colostrum of non-smoking (2.43 ± 0.70 nM/min/mg) and second-hand smoking (2.81 ± 1.09 nM/min/mg) (Figure 9).

Figure 9. Comparison of GPx concentration (nM/min/mg of protein) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). a – statistically significant difference compared to the group of non-smoking women (p ≤ 0.0001). b – statistically significant difference compared to the group of women exposed to SHS (p ≤ 0.0001). j – statistically significant difference compared to the group of non-smoking women (p = 0.0013). k – statistically significant difference compared to the group of women exposed to SHS (p = 0.0013). l – statistically significant difference compared to the group of non-smoking women (p = 0.0002). m – statistically significant difference compared to the group of women exposed to SHS (p = 0.0002). * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.2.9. Superoxide dismutase Statistically significant differences of the determined SOD concentrations were demonstrated in the case of colostrum in smoking women (634.83 ± 292.13 nM/min/mg) and women exposed to second-hand tobacco smoke (429.28 ± 246.62 nM/min/mg) compared to non-smoking women (328.00 ± 122.40 nM/min/mg). A significantly higher level of SOD activity was also noted for mature milk of smoking women (699.40 ± 263.91 nM/min/mg) compared to non-smoking (538 ± 162.55 nM/min/mg) (Figure 10).

16

Figure 10. Comparison of SOD concentration (nM/min/mg of protein) in plasma, colostrum, and mature milk of study groups (blood and colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). a – statistically significant difference compared to the group of nonsmoking women (p ≤ 0.0001). n – statistically significant difference compared to the group of nonsmoking women (p = 0.0225). * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.2.10. Catalase The activity of CAT was significantly higher in colostrum of tobacco smoking patients (1541.81 ± 729.00 nM/min/mg) compared to patients exposed to SHS (1212.00 ± 750.42 nM/min/mg) as well as in the SHS group compared to the nonsmoking group (1014.57 ± 684.84 nM/min/mg) (Figure 11). In mature milk of tobacco smoking women’s group, the activity of CAT (3652.23 ± 1669.22 nM/min/mg) was significantly higher than that observed in the non-smoking group (1997.00 ± 866.00 nM/min/mg) and SHS group (2264.59 ± 1374.19 nM/min/mg).

Figure 11. Comparison of CAT concentration (nM/min/mg of protein) in colostrum, and mature milk of study groups (colostrum samples collected 1 ± 2 days after delivery; mature milk samples collected 30 ± 7 days after delivery). 17

a – statistically significant difference compared to the group of non-smoking women (p ≤ 0.0001). b – statistically significant difference compared to the group of women exposed to SHS (p ≤ 0.0001). o – statistically significant difference compared to the group of non-smoking women (p = 0.0015). p – statistically significant difference compared to the group of women exposed to SHS (p = 0.0015). * – statistically significant difference compared to the result observed for colostrum in the same group (p < 0.05)

3.3. Correlation between cotinine and oxidative stress parameters The significant (p < 0.05), positive correlation (RS = 0.43) was observed between the cotinine and TAEC concentration in the colostrum of tobacco smoking women (Figure 12).

Figure 12. Correlation (RS = 0.43; p < 0.05) between the concentration of cotinine and TAEC in the colostrum of tobacco-smoking women (n = 28).

Moreover, in mature milk of tobacco-smoking women, was observed a negative correlation (RS = -0.52) between the concentration of cotinine and GST activity; p < 0.05 (Figure 13). In other cases, correlations were not observed.

Figure 13. Correlation (RS = -0.52; p < 0.05) between the concentration of cotinine and activity of GST in mature milk of tobacco-smoking women (n = 30). 18

4. DISCUSSION The US Surgeon General, the American Academy of Pediatrics, and the World Health Organization have recommended exclusive breastfeeding for infants until the end of the sixth month after birth life (CDC, 2011b; WHO, 2002; AAP, 2005). The unique composition of human milk provides all of the nutrients necessary for normal development of an infants. There is some controversy regarding whether women who smoke tobacco should breastfeed and how tobacco smoke effects the properties of breast milk (Napierała et al., 2016). Over 5300 chemical compounds have been identified in tobacco smoke. Seventy of these are known carcinogens (Talhout et al., 2011; IARC, 2012). Several studies have demonstrated that many of these chemicals pass into breast milk including nicotine (Napierała 2017). Nicotine contained in breast milk is absorbed in the infant's digestive tract then metabolized in the liver to cotinine (Labrecque et al., 1989). The concentration of cotinine in maternal serum and breast milk is proportional to the number of cigarettes smoked per day by women (Woodward et al., 1986; Mascola et al., 1998). The US Surgeon General, the American Academy of Pediatrics, and the World Health Organization have recommended exclusive breastfeeding for infants until the end of the sixth month after birth life (CDC, 2011b; WHO, 2002; AAP, 2005). The unique composition of human milk provides all of the nutrients necessary for normal development of an infants. There is some controversy regarding whether women who smoke tobacco should breastfeed and how tobacco smoke effects the properties of breast milk (Napierała et al., 2016). Over 5300 chemical compounds have been identified in tobacco smoke. Seventy of these are known carcinogens (Talhout et al., 2011; IARC, 2012). Several studies have demonstrated that many of these chemicals pass into breast milk including nicotine (Napierała 2017). Nicotine contained in breast milk is absorbed in the infant's digestive tract then metabolized in the liver to cotinine (Labrecque et al., 1989). The concentration of cotinine in maternal serum and breast milk is proportional to the number of cigarettes smoked per day by women (Woodward et al., 1986; Mascola et al., 1998). In this study, we observed a significant positive correlation (p<0.05) between the serum cotinine concentration and colostrum among lactating women initiating lactation in both the group of smoking and second-hand smoke exposed women (RS=0.90, n=28; RS=0.84, n=18, respectively). In our study, tobacco smoking and number of cigarettes smoked, or exposure to second-hand smoke, or non-smoking and lack of exposure was documented by using a questionnaire and confirmed by measuring cotinine levels in maternal serum. Among some women, despite declaration of not smoking status, the concentration of cotinine (nicotine metabolite) was observed at a level comparable to those associated with tobacco smoking. These women also did not declare nicotine replacement therapy or e-cigarettes use. These patients were included in the group of smokers. Nicotine can also be present in tea, tomatoes, cauliflower and potatoes but it has not been proven that in the concentration that can be determined in the biological material after ingestion. Some earlier studies have shown that part of pregnant women or young mothers want to hide the fact of smoking tobacco due to social norm pressures, as is also indicated by the results of this study (Kim, 2016). While commonly used biomarker to validate self-reported smoking status is cotinine, the selection of an optimal cotinine cutoff value for distinguishing true smokers from true nonsmokers brings many difficulties. The Kim (2016) assumed 104 articles, 32 of which provided sensitivity and specificity of a cotinine cutoff value and 19

determination methods for the given cutoff value (Kim, 2016). The serum cotinine cutoff of 10–20 ng/mL, have been commonly used to validate self-reported smoking status. Nonsmokers exposed to typical levels of SHS had less serum cotinine levels than pointed cutoff. However, it has been found that the ratio of cotinine per cigarette smoked during pregnancy was much lower than the ratio after pregnancy (3.53 ng/mL vs. 9.87 ng/mL of salivary per cigarette) (Rebagliato et al., 1998). This suggest, that the available cutoff value for adult pregnant populations may be not applicable to the general population. Therefore, obtained in this study mean level of cotinine 18.06 ng/mL in the serum of patients included in the group exposed to second hand tobacco smoke (based on their declaration), may indicate that some of the women could have been occasional smokers as well. In future studies, more attention should be paid to estimating the cut-off for cotinine for pregnant women smoking tobacco. Moreover, previous studies have confirmed that a large proportion of women who smoked during pregnancy or those who transiently quit smoking return to smoking during lactation (Ashford, 2009; Napierała et al., 2016). In our study we observed that one month after delivery, about 16% of women who did not smoking during the pregnancy, started smoking again. Human milk has a protective effect against excessive oxidative stress. Tobacco smoke results in alterations in these protective effects by disturbing the antioxidativeprooxidative balance of milk with reduced vitamin C levels (average for mature milk: 241.3 +/-293.1 um/L in smokers vs 496.1+/- 325.6 um/L among non-smoking mothers) (Ortega et al., 1998a), as well as reduced vitamin E levels (average in mature milk: 1.79 ± 0.37 mmol/L vs. 2.30 ± 0.77 mmol/L in the control group; p < 0.05) (Ortega et al., 1998b). Moreover, a reduction of total antioxidant status (TAS) in the colostrum of mothers who smoke (p=0.006) (Zagierski et al., 2012). Our study selected markers of oxidative stress and the activity of antioxidative enzymes in mother's blood and colostrum sampled one day after delivery (± 2 days) and in the mature milk sampled 30 (+/- 7 days) after birth. During lactation, there are changes in the protein content of breast milk and these previously reported changes were documented in our study, with 30% lower protein content in mature milk compared to colostrum (p<0.05). Tobacco smoke can also reduce the total plasma protein concentration (Napierała et al., 2017). Shamsi et al reported lower total protein concentrations in the colostrum of mother's exposed to second-hand smoke compared to non-smoking women (Shamsi et al., 2015). Bachour et al. found total protein concentration 12% less in the tobacco smokers than in non-smokers mature milk (Bachour et al., 2012). There are large amounts of ROS in tobacco smokes that may cause oxidative damage to milk proteins reducing their biologic activity. Further, the reduction in total protein content reduces the nutritional value of milk for the infant and specially the infant's immune system (Ballard and Morrow, 2013). However, in our own studies the mean concentration of total protein between study groups was comparable. Vitamins A, E and C, B-carotene, albumin, bilirubin, cysteine, coenzyme Q, lactoferrin and antioxidant enzymes SOD, CAT, CPx are antioxidants that play a significant role in the total antioxidant status (TAS) in infants. The concentration of some of these antioxidants in breast milk are dependent on maternal diet, vitamin supplementation, and place of residence. Dietary history of mother's participating in our study did not reveal dietary deficiencies for a reduced breast milk TAC. However, it has been previously shown (Zagierski et al 2012) that maternal smoking is one of the most significant factors in reducing TACs in both plasma and human milk. We 20

found that TAS expressed as Trolox equivalents (TEAC) was significantly reduced in both plasma and colostrum of mothers who smoked tobacco (plasma reduced by 25% and colostrum by 15.5%) or who were exposed to second-hand tobacco smoke (19% and 25% respectively for smokers) compared to non-smokers (p<0.0001). These findings have been reported by others (Ermis et al., 2005; Zagierski et al., 2012). Lower total antioxidative potential in colostrum sampled one day after childbirth (± 2 days) can be caused by greater oxidative stress observed during pregnancy and additionally increased by labour (Ademuyiwa et al., 2007). S-nitrosylation is the covalent attachment of nitrogen monoxide group to the thiol side chain of cysteine which is important for post-translational regulation of most classes proteins (Hess et al., 2005). However, enhancement of RNS due to excessive production of toxic nitric oxides can lead to the posttranslational modification of the cysteine in proteins. Existing evidence suggests that those mechanisms can lead to functional disruption of proteins, mitochondrial dynamics, protein folding in the ER or transmission of signals contributing to synaptic damage, neurodegeneration, and even cell death (Napierała et al., 2017). We found that RSNO in the serum of tobacco smoking women to be increased by 21%, similar to the group of women exposed to second-hand smoke compared to non-smoking mothers. This unique observation requires further study; however, higher levels of reactive nitrogen species have been reported among women with pre-eclampsia and hypercholesterolemia (Rossi et al., 2001). Deregulation of protein S-nitrosylation has been associated with a higher risk for chromosomal damage, and in patients with Parkinson's diease, multiple sclerosis, pulmonary hypertension, asthma, and cystic fibrosis (Hess et al., 2005; Pietryga et al., 2017). Nitric oxide has both prooxidative and antioxidant properties. We found significant elevations of NO in plasma and colostrum from women who smoked tobacco compared to non-smokers (28.5% for plasma and 76% for colostrum) and similar elevations also in mature milk. By reacting with free radicals, NO inhibits lipid peroxidation and in the presence of 02-, NO is converted to peroxynitrite (ONOO-), a strong biological oxidant. The formation of ONOO- intensifies when the SOD activity is low (Hess et al., 2005; Bartosz, 2013). As a result of SOD deficiency, in the case of the continuous oxidative stress, neuronal cell death and early development of cancer occurs (Bartosz, 2013). Ermis et al. demonstrated that SOD activity is reduced in colostrum (7th day after delivery) of tobacco smoking women (p = 0.015) and women exposed to SHS (p = 0.004) compared to the control group (by 41.40% and 38.20%, respectively) (Ermis et al., 2005). In consequence, excessive production of ONOO- can occur in the milk of actively or second hand tobacco smoking mothers and contribute to intensified free radical processes damaging cellular biomolecules, proteins in particular (O'Donnell and Freeman, 2001). However, the results of our studies did not confirm this theory, as a significantly higher SOD concentration was observed in the colostrum of actively smoking mothers (by 93.55%) and mothers exposed to SHS (by 30.88%) compared to non-smoking mothers, which may point to adaptive changes in the properties of milk antioxidant barriers in response to increased oxidative stress. Moreover, SOD level was significantly higher in mature milk than in colostrum, which might reflect the various antioxidant needs during development of newborns. Our findings have been collaborated by others (L'Abbe and Friel, 2000). Numerous reports indicate that increased concentration of MDA and other products of peroxidation of thiobarbituric acid reactive substances (TBARS) is an important marker of oxidative stress in biological material (plasma, saliva) sampled from 21

smoking individuals (Lykkesfeldt, 2007). In agreement with others (Ermis et al., 2005) we also found increased concentrations of peroxidation products, in the colostrum and mature milk of women who smoke compared to non-smokers (p<0.05) (See Figure 6). Glutathione peroxidase (GPx), an enzyme that catalyzes the reduction of lipid hydroperoxides and aldehydes by coupling them to glutathione would found to be significantly higher in the plasma and mature milk of smoking women than in the non-smoking group (by 28.27% and by 48.44%, respectively) and the group exposed to second hand tobacco smoke (by 22.08% and by 48.44%, respectively). We observed an inverse relationship in the case of colostrum. Increased GPx reflects the physiologic response to ongoing lipid peroxidation in milk. Similarly we found significant differences of glutathione S-transferase activity in colostrum and mature milk of women who smoked compared to our non-smoking mothers in both colostrum and mature milk. So far, the available literature provides no information on changes in glutathione S-transferase activity in breast milk depends of the stages of lactation and tobacco smoke exposure on this enzyme’s activity in milk. Glutathione S-transferase (GST) play a major role in cellular detoxification against some xenobiotics, carcinogens as well as against oxidative stress. Therefore, it is believed that this enzyme may play a key role in the regulation of susceptibility to some cancers (Beckett and Hayes, 1993; Bartosz, 2013). Endogenous tripeptide glutathione is involved in detoxification of electrophilic xenobiotics through the formation of S-conjugates with toxic metabolites in the second phase of biotransformation using GST. GSH forms among other S-conjugates with products of lipid peroxidation. Moreover, GST can protect proteins from the effect of oxidative stress, through modulation of their function by S-glutathionylation of cysteine residues (Allocati et al., 2018). In ours study we observed a negative correlation (RS = -0.52) between GST activity and cotinine concentration in mature milk (p < 0.05). Glutathione S-transferase is involved in biotransformation of others compounds of tobacco smoke for example acrolein (Allocati et al., 2018). However, GST lower activity, depending on the size of the exposure and altered cellular redox homeostasis in the presence of tobacco smoke, may indicate the impairment of the detoxification and antioxidant mechanisms involving GST. Tobacco smoke can affect the expression of genes encoding enzymes responsible for detoxification, such as GST, and thereby increase the activation of environmental toxins. In contrast, an increased TEAC concentration in the colostrum depending on the concentration of cotinine among smoking mothers (RS = 0.43; p < 0.05) may indicate the activation of compensation mechanisms related to exposure to tobacco smoke and the imbalance of redox homeostasis. Some reports indicate that active smokers produce compensatory blood levels of GSH that effectively prevent peroxidation of plasma lipids. Thus, somehow smokers can fight with oxidative stress via increasing the production of glutathione or its release into the plasma. However, our in study did not prove differences in the plasma glutathione concentration between the study groups. Beyond the increased production of GSH, the increased TEAC could be observed due to the release of intracellular antioxidants into the plasma. Moreover TEAC higher concentration among smokers may be also connected with activation of their immune system against the tobacco smoke compounds. On the other hand, increased oxidative stress following exposure to the tobacco smoke in some cases may overcome the immune system and cause oxidative damage to biomolecules (Motalebnejad et al., 2013). 22

Catalase also is crucial against oxidative damage caused by ROS by degrading surplus H202. The decomposition of hydrogen peroxide takes place mainly with the participation of GPx, so catalase has only auxiliary functions and shows activity in the event of extremely high H2O2 concentrations (He et al., 2008). Therefore, longterm exposure to tobacco smoke results in increased CAT activity to prevent excessive production of hydroxyl radicals associated with the Fenton reaction (Bartosz, 2013). We found concentrations of CAT to be significantly higher in the colostrum and mature milk of tobacco smoking women and those exposed to second-hand smoke compared to non-smoking mothers (p <0.05). The results of our studies demonstrate clearly that tobacco smoke is a significant factor increasing the oxidative stress in human milk of mothers who smoke and lesser so in mothers exposed to second-hand smoke by lower TEAC and higher TBARS concentrations. The increased defense system of colostrum and mature milk in response to ROS generated by tobacco smoke is evidenced by an increased in the activity antioxidant enzymes (SOD, GST, GPx, and CAT). However, as has been shown by the demonstrated correlations with cotinine concentration, this may depend of the amount, duration as well as frequency of exposure to the tobacco smoke. Therefore, the authors of the article point out that in the future studies with a larger number of patients, it would be advisable to consider the evaluation of oxidative stress biomarkers depending on the magnitude of exposure, for example based on intervals of the number of cigarettes smoked per day. The innate role of human milk as a defense against oxidative stress is substantially reduced when a mother smokes tobacco or is exposed to second-hand smoke. The best form to stop the progression of these processes would be to stop smoking as well as protect women during pregnancy and lactation from second-hand tobacco smoke exposure. Former smoking was not found to alter TP, TAC and MDA in the colostrum milk (Poniedziałek et al., 2018). Greater public awareness (especially among medical personnel, future mothers, their relatives and other people from their surroundings) about the adverse effects of tobacco smoking during lactation on breast milk quality and its protective effects is urgently needed. 5. CONCLUSIONS:  Awareness among women of the harmful effects of smoking during lactation is lower than the public health warnings about smoking during pregnancy. Our study should be a stimulus for greater public health awareness of the adverse effects of tobacco smoking during lactation on breast milk quality and its protective effects  Among some women declaring non-smoking, cotinine was found in blood serum, which confirms the need to verify questionnaire data by measuring the exposure biomarker to document actual tobacco or nicotine use.  Tobacco smoking is significant factor increasing oxidative stress in mother’s plasma, colostrum and mature milk manifested among other by lower TEAC and higher TBARS concentration.  There are increased activities of antioxidant defenses in colostrum and mature milk of women in response to ROS generated by tobacco smoke, manifested by an increase in the activity of antioxidant enzymes (SOD, GST, GPx and CAT). However, as suggest the demonstrated correlation with cotinine concentration, this may depend of the amount of exposure to the tobacco smoke.  Exposure to tobacco smoke by mothers during lactation results in changes to the protective effects of both colostrum and mature milk and may result in the mitigation of the beneficial effects of breastfeeding on infant health and well-being. 23

ACKNOWLEDGMENTS We thank all study participants, research staff, and students who participated in this work. This work was supported by the resources intended to maintain research potential in Poznan University of Medical Sciences, Poznan, Poland (2014-2016: 502-1403315431-41132 and 2016-2017: 502-14-03315431-10554), and in Loma Linda University, Loma Linda, California (845-0331-5665-04648). FUNDING SOURCES This work was supported by the resources intended to maintain research potential in Poznan University of Medical Sciences, Poznan, Poland (2014-2016: 502-1403315431-41132 and 2016-2017: 502-14-03315431-10554), and in Loma Linda University, Loma Linda, California (845-0331-5665-04648). AUTHORS' CONTRIBUTIONS E.F., T.A.M. and M.N. designed the study. E.F., T.A.M. and M.N. managed the funding. E.F. and M.N. managed the project. M.N., K.M. and J.M. conducted selection of patients, collected clinical and epidemiological data and samples of biological material. M.N. performed laboratory measurements of samples. M.N. and I.M. performed data preprocessing, quality controls and statistical analysis. M.N. analyzed the data, wrote the first draft and made major revisions to the manuscript. E.F., A.T.M, J.M. made major comments/edits to the manuscript. All authors made contributions in commenting and revising the paper. BIOETHICS COMMITTEE APPROVAL The study protocol was approved by the Bioethics Committee under Act 593/13 of 13 June 2013, based on Polish legislation and Good Clinical Practice at the Poznan University of Medical Sciences. REFERENCES [1] AAP (American Academy of Pediatrics): Gartner LM, Morton J, Lawrence RA, Naylor AJ, O'Hare D, Schanler RJ, Eidelman AI (Section on Breastfeeding). 2005. Breastfeeding and the use of human milk. Pediatrics. 115(2):496-506. PMID: 15687461 DOI: 10.1542/peds.2004-2491 [2] Ademuyiwa O, Odusoga OL, Adebawo OO, Ugbaja R. 2007. Endogenous antioxidant defences in plasma and erythrocytes of pregnant women during different trimesters of pregnancy. Acta Obstet Gynecol Scand. 86(10):11751182. PMID: 17851796 DOI: 10.1080/00016340701515357 [3] Albers L, Sobotzki C, Kuß O, Ajslev T, Batista RF, Bettiol H, Brabin B, Buka SL, Cardoso VC, Clifton VL, Devereux G, Gilman SE, Grzeskowiak LE, Heinrich J, Hummel S, Jacobsen GW, Jones G, Koshy G, Morgen CS, Oken E, Paus T, Pausova Z, Rifas-Shiman SL, Sharma AJ, da Silva AA, Sørensen TI, Thiering E, Turner S, Vik T, von Kries R. 2018. Maternal smoking during pregnancy and offspring overweight: is there a dose-response relationship? An individual patient data meta-analysis. Int J Obes (Lond). PMID: 29717267 DOI: 10.1038/s41366018-0050-0 [4] Allocati N, Masulli M, Di Ilio C, Federici L. 2018. Glutathione transferases: substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases. Oncogenesis. 7(1):8. doi: 10.1038/s41389-017-0025-3. 24

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Awareness regarding the harmful effects of smoking during lactation is low.

•

The questionnaire data about smoking status should be confirmed by biomarker evaluation.

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Tobacco smoke significantly increase TBARS and decrease TEAC in colostrum and mature milk.

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ROS generated by tobacco smoke increase the activity of antioxidant enzymes (SOD, GST, GPx, CAT).

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Tobacco smoke exposure during lactation may influence infant and child health.

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