Antihypertensive and antioxidant effects of dietary black sesame meal in pre-hypertensive humans

Jatuporn Wichitsranoi
Natthida Weerapreeyakul
Patcharee Boonsiri
Chatri Settasatian
Nongnuch Settasatian
Nantarat Komanasin
Suchart Sirijaichingkul
Yaovalak Teerajetgul
Nuchanart Rangkadilok
Naruemon Leelayuwat
Antihypertensive and antioxidant effects of dietary black sesame meal in pre-hypertensive humans
R E S E A R C H Open Access
Background: It has been known that hypertension is an independent risk factor for cardiovascular disease (CVD). CVD is the major cause of morbidity and mortality in developed and developing countries. Elevation of blood pressure (BP) increases the adverse effect for cardiovascular outcomes. Prevention of increased BP plays a crucial role in a reduction of those outcomes, leading to a decrease in mortality. Therefore, the purpose of this study was to investigate the effects of dietary black sesame meal on BP and oxidative stress in individuals with prehypertension. Methods: Twenty-two women and eight men (aged 49.8 ± 6.6 years) with prehypertension were randomly divided into two groups, 15 subjects per group. They ingested 2.52 g black sesame meal capsules or placebo capsules each day for 4 weeks. Blood samples were obtained after overnight fasting for measurement of plasma lipid, malondialdehyde (MDA) and vitamin E levels. Anthropometry, body composition and BP were measured before and after 4-week administration of black sesame meal or a placebo.Results:The results showed that 4-week administration of black sesame meal significantly decreased systolic BP (129.3 ± 6.8 vs. 121.0 ± 9.0 mmHg, P < 0.05) and MDA level (1.8 ± 0.6 vs. 1.2 ± 0.6 μmol/L, P < 0.05), and increased vitamin E level (29.4 ± 6.0 vs. 38.2 ± 7.8 μmol/L, P < 0.01). In the black sesame meal group, the change in SBP tended to be positively related to the change in MDA (R = 0.50, P = 0.05), while the change in DBP was negatively related to the change in vitamin E (R = -0.55, P < 0.05). There were no correlations between changes in BP and oxidative stress in the control group. Conclusions: These results suggest the possible antihypertensive effects of black sesame meal on improving antioxidant status and decreasing oxidant stress. These data may imply a beneficial effect of black sesame meal on prevention of CVD.

Background

Hypertension is an important risk factor for cardiovascular disease (CVD) in developing countries. Elevation of blood pressure (BP) is a risk factor for adverse cardiovascular outcomes, including stroke, myocardial infarction, renal failure and death [1]. Prevention of increased BP therefore plays a crucial role in a reduction of those outcomes. Impaired balance between relaxing and contracting factors in the endothelium of blood vessels is an important pathogenic mechanism of hypertension. Increased pro-oxidant and decreased antioxidant activities have been shown to be some of the mechanisms of the pathogenesis of hypertension [2].

It has been reported that sesame seeds can improve oxidative stress due to actions of their contents of vitamin E and lignans including sesamin, sesamolin and sesamol [3][4][5][6][7]. Thus, sesame is likely to have potential health benefits in relation to CVD by its antihypertensive effects [8][9][10][11]. Although many previous studies have shown different effects of vitamin E on BP [12][13][14], these studies investigated the effect of supplementation of either alphatocopherol alone or mixed with gamma-tocopherol on BP in diabetic [12,13] or hypertensive [14] patients who took antihypertensive drugs. Interactions between alpha-tocopherol and the drug may diminish the antihypertensive effect on BP [15]. Moreover, the dose of vitamin E in those previous studies may have been so high that it caused increased or unchanged effects on BP.

Interestingly, the antihypertensive effect of black sesame (Sesamum indicum Linn.) meal, a product of sesame oil manufacturing, does not seem to have been previously investigated. Positive results may provide additional value to the manufactured product. Since we wanted to examine preventive effects, we performed the research on pre-hypertensive [16] healthy individuals who did not take any medicine; hence the vitamin E could exercise its antioxidant activity without interference with any antihypertensive drug. Moreover, black sesame meal contains gamma-tocopherol which was reported to be lower in patients with coronary heart disease than control subjects [17]. Taken together with the safety dose of vitamin E in black sesame meal in this study [18], this may reveal an anti-hypertensive effect of black sesame meal.

Based on knowledge of the effects of sesame seed and lignans on oxidative stress, which is one of mechanisms of the pathogenesis of hypertension [2], it was hypothesized that black sesame meal may have an antihypertensive effect in pre-hypertensive humans via improving antioxidant status and decreasing oxidant stress. Therefore, the aim of this study was to investigate the effects of 4-week administration of black sesame meal on BP and oxidative stress in pre-hypertensive humans.

Methods

Study design

A double-blind, placebo-controlled investigation was undertaken. Subjects were divided into two groups, with 11 men and 4 women in each group, matched by age, BMI and BP: a black sesame meal group (SG) (N = 15, aged 49.3 ± 7.7 years), and a placebo group (PG) (N = 15, aged 50.3 ± 5.6 years). Subjects and investigators were blinded as to the composition of the black sesame meal and placebo capsules. After being screened by physical and blood examination, all subjects were asked to complete health questionnaires in order to provide health information including cardiovascular risk factors. Subjects were seen on two visits: before and after 4-week administration of either black sesame meal capsules or placebo capsules. During the 4 weeks, subjects were asked to take 6 capsules each time, three times a day, with water after a meal. They were asked to avoid vitamins and other dietary supplements during the administration period. Subjects were instructed not to change their diet and exercise routine throughout the trial. At both visits, after a 12-h fasting period, blood samples were collected to measure blood parameters. Anthropometry (height, weight, and waist and hip circumferences), body composition and BP were also measured.

Subjects

Thirty middle-aged subjects (22 men and 8 women) were recruited from the general population, supported by an annual health checkup program performed at the Faculty of Associated Medical Sciences, Khon Kaen University, during 2008-2009. Subjects had no other diseases except prehypertension as indicated either by systolic blood pressure (SBP) from 120 to 139 mmHg or diastolic BP (DBP) from 80 to 89 mmHg [16]. The patients were not currently taking any medication or dietary supplementation that affected BP. This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving humans were approved by the Ethics Committee of Khon Kaen University (HE 510254). Written and verbal informed consent was obtained from all subjects; verbal consent was witnessed and formally recorded.

Power calculation

A change in SBP after the ingestion of black sesame meal was used to calculate the sample size of this study [9]. The magnitude of the change in SBP was 5 mmHg for the power calculation. It was decided to require 90% power at a significance level of 0.05. Thus, having at least 15 subjects was required to finish this study.

Preparation of black sesame meal

The black sesame meal was prepared at the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand. The sesame seeds were roasted before being pressed. The remaining sesame meal was grounded into powder and mixed with an adsorbent. This process is the same as that used for commercial preparation. Each capsule of black sesame meal was formulated to contain 0.42 g of black sesame meal. The contents (mean ± SD) of carbohydrate, protein and fiber of the black sesame meal were 46.37%, 21.57% and 14.12%, respectively. Moreover, sesamin and sesamolin contents were 1.172 ± 0.002 mg/g DW and 0.605 ± 0.003 mg/g DW, respectively [19]. Total tocopherol was 105.41 ± 2.49 μg/g DW, mostly consisting of gamma-tocopherol (102.78 ± 2.46 μg/g DW); alpha-tocopherol was not detectable [19]. The placebo capsule contained the same contents, but without any black sesame meal. Both capsules were the same shape and color.

Measurement of BP

BP (mmHg) and heart rate (HR,/min) were measured in the morning, after a 20-min rest, using an automatic sphygmomanometer (Accurtorr 1A; Datascope, Japan) on the right upper arm while the subject was in a sitting position. Average BP and HR were calculated from three measurements after an almost stable BP seemed to have been reached. SBP and DBP were used to calculate mean arterial pressure (MAP) by the formula: MAP = DBP + 1/3 (SBP -DBP).

Measurement of malondialdehyde (MDA)

A plasma lipid peroxidation marker, MDA, was estimated using thiobarbituric acid, as previously described [20]. In brief, 150 μl of plasma was reacted with 125 μl 10% trichloroacetic acid, 125 μl 5 mM ethylenediaminetetraacetic acid, 125 μl 8% sodium dodecyl sulfate, and 10 μl 0.5 μg/ml butylated hydroxytoluene. After vortexing vigorously for 30 s, the mixture was incubated for 10 min at room temperature. Then 500 μl 0.6% thiobarbituric acid was added, and the mixture was heated at 95°C for 30 min. After cooling to room temperature, the mixture was centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm using a spectrophotometer (Genesys 20, SN: 35 gk 130009; Thermo Fisher Scientific, USA). A standard curve was generated with appropriate concentrations of 1,1,3,3-tetraethoxypropane standard (0.3-10 μmol/L); the plasma MDA concentration was expressed as μmol/L of plasma.

Measurement of vitamin E

Serum vitamin E was determined using high performance liquid chromatography (HPLC), according to the method of Thurnham [21], by injection into a reversed-phase C-18 Spherisorb ODS2 column (diameter 5 μm, 4.6 × 100 mm; Waters, USA). The mobile phase consisted of methanol/ acetonitrile/dichloromethane at a ratio of 4:4:1 with a flow rate of 1 mL/min. A wavelength UV-visible detector (model 2847, Waters) was set at a wavelength of 292 nm for the detection of vitamin E. Quantification was based on peak-height measurement, using the Clarity 22.214.171.124 software program, version C22 (DataApex, Czech Republic).

Statistical analysis

All statistics were generated using Stata statistical software, version 10 (StataCorp, USA). Data were expressed as means ± SD. Changes in BP, MDA and vitamin E levels between groups were compared by analysis of covariance (ANCOVA), with adjustment for baseline values. The variables under study before and after the administration within groups were compared by Student's paired t-test. The relationships between changes in oxidative stress variables and BP were analyzed by Pearson's correlation. Statistical analyses were two-sided. If the statistical probability (P -value) was less than 0.05, the differences were considered to be statistically significant.

Results

Baseline characteristics of subjects are shown in Table 1. All subjects were pre-hypertensive individuals. No significant differences in age, anthropometry, body composition, and levels of MDA and vitamin E, SBP, DBP, MAP and HR were found between SG and PG groups.

In the SG group, after 4 weeks of administration of black sesame meal, SBP was significantly decreased when compared with before administration (121.0 ± 9.0 vs. 129.3 ± 6.8 mmHg, P < 0.05) ( Figure 1A). Meanwhile, in the PG group, SBP was slightly decreased after the placebo administration when compared with before administration values (130.4 ± 4.8 vs. 130.6 ± 9.5 mmHg, P = 0.52). The decrease in SBP in the SG group was significantly greater than that in the PG group (P < 0.05) with adjustment for baseline values. There were no apparent side effects induced by black sesame meal throughout the study.

In the SG group, after 4 weeks of administration of black sesame meal, DBP was decreased when compared with before administration (72.8 ± 9.8 vs. 77.0 ± 7.4 mmHg, P = 0.20) ( Figure 1B). Meanwhile, in the PG group (85.9 ± 12.1 vs. 80.6 ± 7.7 mmHg, P = 0.14), where the level was higher than before administration. However, the changes in DBP between groups with adjustment for baseline values were not significantly different. Moreover, there was no significant difference in changes of average HR with adjustment for baseline values in both PG and SG subjects (66.8 ± 7.4 and 68.7 ± 9.1/min before and 71.2 ± 11.1 and 71.4 ± 6.4/min after 4-week administration of black sesame meal and placebo, P = 0.76).

Plasma MDA concentrations significantly decreased after the 4-week administration of black sesame meal when compared with before administration concentrations (1.2 ± 0.6 vs. 1.8 ± 0.6 μmol/L, P < 0.05). There were no significant differences in MDA concentrations, however, before and after 4-week administration of a placebo (1.8 ± 0.5 vs. 1.9 ± 0.6 μmol/L, P = NS). When compared with the PG subjects, the SG subjects had significantly lower plasma MDA concentrations after the 4-week administration with adjustment for baseline values (1.8 ± 0.5 (PG), 1.2 ± 0.6 (SG) μmol/L, P < 0.05) ( Figure 2).

As shown in Figure 3, plasma vitamin E (total tocopherol) concentrations after 4-week administration of black sesame meal were significantly increased when compared with before administration concentrations (38.2 ± 7.8 vs. 29.4 ± 6.0 μmol/L, P < 0.01). There were no significant differences in vitamin E concentrations before and after 4-week administration of the placebo (29.8 ± 6.0 vs. 26.5 ± 5.5 μmol/L, P = NS). When compared with the PG group, the SG group had significantly greater plasma vitamin E concentrations after 4-week of   Only in the black sesame meal group did the change in SBP tend to be positively related to the change in MDA concentration (R = 0.50, P = 0.05), while the change in DBP was negatively related to the change in vitamin E concentration (R = 0.55, P < 0.05). There were no significant correlations between changes in BP and oxidative stress indicators in the placebo group.

Discussion

This study appears to be the first to reveal the possible antihypertensive effects of black sesame meal in participants with prehypertension. The results demonstrated that after matching for age, BMI and BP, 4-week daily administration of 2.52 g black sesame meal caused a significant reduction in SBP, by an average of 8.2 mmHg.

Interestingly, the INTERSALT study revealed that a reduction of 2 to 3 mmHg of SBP was associated with a 4% decrease in mortality from CVD in the USA and UK [22] and a 6.4% decrease in mortality from cerebral vascular disease in Japan [1]. Based on these longer term studies, if the present reduction in BP with sesame meal (8.2 mmHg) was sustained in the long term, this could reduce the risk of CVD and stroke by 16.4% and 26.2% respectively. This study shows that the ingestion of black sesame meal may have a potential effect on reduction in mortality from CVD and stroke.

Although no previous study has directly investigated the effect of black sesame meal on BP, many studies have reported the potential antihypertensive effect of sesame's contents (i.e. lignan and vitamin E) in humans and rats [8][9][10][11]23]. However, this is inconsistent with many other previous studies which reported different results [12][13][14]. Some previous studies have reported that supplementation of either alpha-tocopherol alone or mixed with gammatocopherol increased [12] or did not change [13] BP in diabetic or treated hypertensive patients [14]. This may be attributed to an interference of alpha-tocopherol and the antihypertensive drug taken by some subjects in previous studies; whereas subjects in this study were healthy and did not take any antihypertensive drug. Moreover, the dose of vitamin E in those previous studies may have been so high that it caused increased or unchanged effects on BP.

A possible mechanism responsible for the antihypertensive effect of black sesame meal in this study may be an improved balance between relaxing and contracting factors in the endothelium of blood vessels. In this study, black sesame meal may have enhanced the relaxing factor, resulting in improved endothelium-dependent vasorelaxation in the pre-hypertensive participants relating to oxidative stress. The antihypertensive effect of sesamin, in mildly hypertensive humans was supported by a previous study [9]. The authors reported that the ingestion of 60 g of sesamin per day for 4 weeks decreased SBP by an average of 3.5 mmHg, and DBP by 1.9 mmHg [9]. Moreover, the increased plasma vitamin E in this study may be due to the increased vitamin E or the inhibition of catabolism of vitamin E. In addition, bioavailability of vitamin E may be increased by interactions between sesame lignan and tocopherols [24]. The accumulation of vitamin E acts by detoxifying the hydroxyl and proxy radicals, leading to reduced lipid peroxidation, or by reducing excess tissue aldehydes [11]. This is supported by many previous studies, in which supplementation of either vitamin E or sesamin and sesamolin demonstrated that these antioxidants inhibit lipid peroxidation [3][4][5][6][7]25]. The antioxidant effect is likely to contribute to the decreased endothelial dysfunction from free radicals [26], resulting in an increase in the vasorelaxing factor, nitric oxide (NO) [27].

However, a mechanism that is not relevant to oxidative stress cannot be ruled out. This was supported by a study by Ward et al. (2004) which failed to show a link between oxidative stress and BP [28]. It was shown that gammatocopherol supplementation increased protein expression of nitric oxide synthase [29], which stimulates vasorelaxation. Moreover, the potential effect of vitamin E on membrane fluidity is likely to be another mechanism of antihypertension. In vitro study, vitamin E was shown to preserve endothelial cell (EC) migration in oxidized lowdensity lipoprotein cells (oxLDL) and restore the endothelial monolayer after injury by inhibiting changes in membrane integrity caused by oxLDL [30]. A previous study in rats demonstrated that antihypertensive rats had lower BP than normotensive rats due to reduced membrane fluidity after ingestion of vitamin E 3 d/week for 3 weeks [31]. These non-antioxidant properties of vitamin E could be important in the prevention of atherosclerosis, resulting in a reduction in BP in humans. Sesamin supplementation also induced NO and inhibited endothelin-1 production by EC [32]. These findings indicate an improved ability of the endothelium to relax. In addition, a recent study has shown that sesamin inhibited some CYP450 enzymes and the production of 20-hydroxyeicosatetraenoic acid (20-HETE), which might influence BP independently of any effects on oxidative stress [33]. Moreover, sesamin increased Ca 2+ antagonistic vasorelaxing activity [34]. It should be emphasized in this discussion that these hypotheses are only on the basis of the decrease in MDA and increased vitamin E, because other possible mechanisms were not measured.

A limitation of this study is a lack of data on endothelium-dependent vasorelaxation determined by NO and other antioxidants such as vitamin C. Moreover, the potential effect of vitamin E on membrane fluidity and the ability of sesame lignans to inhibit 20-HETE synthesis in human renal and liver microsomes were not investigated by this study. Thus, further investigation of these variables explaining the mechanisms of black sesame meal on prevention of hypertension is needed. Importantly, having no discernible side effects from the ingestion of black sesame meal throughout this study implies that it may be safe as a nutritional supplement for health promotion.

Conclusions

This study suggests a beneficial effect of dietary black sesame meal on a reduction in blood pressure in prehypertensive humans. It is likely that the antihypertensive effect is due to decreased oxidative stress. Taken together with the absence of side effects and inexpensive preparation, the regular ingestion of dietary black sesame meal may be beneficial for CVD prevention in individuals with prehypertension, or even those with hypertension. A future study that investigates this advantageous effect is suggested.

Figure 1
1Average levels of SBP (A) and DBP (B) before and after 4-week administration of black sesame meal and a placebo in subjects with prehypertension. Values are expressed as means ± SD. SBP, systolic blood pressure; DBP, diastolic blood pressure; PG, placebo group; SG, black sesame meal group.

Figure 2
2Plasma MDA concentrations before and after 4-week administration of black sesame meal and a placebo in subjects with prehypertension. Values are expressed as means ± SD. PG, placebo group; SG, black sesame meal group; MDA, malondialdehyde.

Figure 3
3Plasma vitamin E concentrations before and after 4week administration of black sesame meal and a placebo in subjects with prehypertension. Values are expressed as means ± SD. PG, placebo group; SG, black sesame meal group.

Table 1
1Characteristics and baseline outcome of the subjects at baselinePG
(n = 15)

SG
(n = 15)

P-value

Age (yr)
50.3 ± 5.6 (39-58)
49.3 ± 7.7 (38-59)
0.70

Height (cm)
161.4 ± 5.4 (154-171) 159.5 ± 7.5 (150-173)
0.45

BW (kg)
66.9 ± 8.7 (53.9-78.5) 68.1 ± 12.1 (57.5-92.7)
0.71

BMI (kg/m 2 )
25.6 ± 2.4 (21.8-29.1) 26.6 ± 3.2 (22.7-34.3)
0.29
%BF
31.5 ± 4.6 (24.7-37.6) 31.6 ± 3.4 (26.7-37.2)
0.92

FM (kg)
21.3 ± 5.1 (13.3-28.5) 21.7 ± 5.2 (15.9-32.7)
0.81

FFM (kg)
45.6 ± 4.7 (37.6-52.9) 46.4 ± 7.6 (37.7-60.9)
0.69

W (cm)
84.6 ± 6.9 (72-94)
85.8 ± 9.6 (74.5-107)
0.66

H (cm)
95.3 ± 4.9 (86-101.5)
96.9 ± 5.4 (88-108)
0.35

W/H ratio
0.89 ± 0.1 (0.77-0.94) 0.88 ± 0.1 (0.79-1.02)
0.75

MDA (μmol/L) 1.9 ± 0.62 (0.86-2.78)
1.8 ± 0.6 (1.19-2.81)
0.67

Vitamin E
(μmol/L)

26.5 ± 5.5 (17.2-34.4) 29.4 ± 6.0 (19.1-45.8)
0.18

SBP (mmHg)
130.4 ± 4.8 (127-138) 129.3 ± 6.8 (121-137)
0.43
DBP (mmHg)
80.6 ± 7.7 (72-99)
77.0 ± 7.4 (65-87)
0.63

MAP (mmHg) 97.2 ± 4.7 (90.3-108.7) 94.4 ± 6.1 (84.7-103.7)
0.51

HR (/min)
66.8 ± 7.4 (59-85)
68.7 ± 9.1 (53-93)
0.61

Values are expressed as means ± SD (minimum-maximum).
PG, placebo group; SG, black sesame meal group; BW, body weight; BMI, body
mass index; %BF, percentage of body fat; FM, fat mass; FFM, fat-free mass; W,
waist circumference; H, hip circumference; W/H ratio, waist and hip
circumference ratio; MDA, malondialdehyde; SBP, systolic blood pressure; DBP,
diastolic blood pressure; MAP, mean arterial pressure; HR, heart rate

Wichitsranoi et al. Nutrition Journal 2011, 10:82 http://www.nutritionj.com/content/10/1/82
AcknowledgementsWe appreciate the assistance of the physical therapists at Queen Sirikit Heart Center of Northeast Thailand. We also appreciate Exercise and Sport Sciences Development and Research Group and Cardiovascular Research Group, Khon Kaen University (a grant number 511010008), Thailand for financial supports. We also would like to thank all subjects for their enthusiastic participation.Authors' contributions JW participated in the design of the study and carried out the plasma MDA and vitamin E assay, physiological measurements and statistical analysis. NW prepared the black sesame meal. PB carried out the vitamin E assay. SS performed the medical cover. NL conceived the study, participated in its design and coordination. NR analyzed vitamin E and lignans in black sesame meal. JW and NL drafted the manuscript. NL is a member of Exercise and Sport Sciences Development and Research Group. NW, CS, NS, NK, SS and YT are members of Cardiovascular Research Group. All authors have read and approved the final manuscript.Competing interestsThe authors are applying for a patent on black sesame meal.
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Assessment of the pro-inflammatory activity of water sampled from major water treatment facilities in the greater Pretoria region

S A Adebayo
Dept of Biomedical Sciences
Faculty of Science
Tshwane University of Technology
Private Bag X6800001PretoriaSouth Africa

L J Shai firstname.lastname@example.com
Dept of Biomedical Sciences
Faculty of Science
Tshwane University of Technology
Private Bag X6800001PretoriaSouth Africa

R Anderson
Dept of Immunology
Faculty of Health Sciences
Medical Research Council Unit for Inflammation and Immunity
University of Pretoria, and Tshwane Academic Division of the National Health Laboratory Service
0001PretoriaSouth Africa

D Du Toit
Dept of Biomedical Sciences
Faculty of Science
Tshwane University of Technology
Private Bag X6800001PretoriaSouth Africa

Dept of Immunology
Faculty of Health Sciences
Medical Research Council Unit for Inflammation and Immunity
University of Pretoria, and Tshwane Academic Division of the National Health Laboratory Service
0001PretoriaSouth Africa

Assessment of the pro-inflammatory activity of water sampled from major water treatment facilities in the greater Pretoria region
C6EF99CBE745EBF8BC613BCC199AD4BB10.4314/wsa.v40i2.20Received 30 January 2013; accepted in revised form 20 March 2014.Endotoxininflammatory activityinterleukin-6mononuclear leukocyteswater quality
Notwithstanding direct detection of microbial/viral pathogens or their associated toxins, the quality of drinking water may also be evaluated according to its pro-inflammatory potential.In this latter setting, contamination with pathogens or their products is determined according to the magnitude of activation of blood-derived immune/inflammatory cells following exposure to test water samples in vitro, usually by monitoring the synthesis of pro-inflammatory cytokines.The primary objective of the current study was to apply this procedure to evaluate the pro-inflammatory potential of water sampled at entry, as well as at various stages of treatment, from 3 major water treatment facilities in the greater Pretoria region, viz., the Daspoort, Hartbeespoort, and Rietvlei Water Treatment Facilities.Control water samples included domestic tap water, bottled water from a commercial source, and distilled water.Peripheral blood mononuclear leukocytes (MNL) were isolated from the blood of healthy, adult, human volunteers (n=3), enumerated, suspended in tissue culture medium RPMI 1640 containing antibiotics at a concentration of 1x10 6 /mℓ, and exposed to the various water samples (10%) for 18 h at 37°C.Following incubation, the cell-free supernatants were assayed for the cytokine, interleukin-6 (IL-6), using a quantitative, sandwich, enzyme immunoassay procedure.The mean values for the untreated control system and for a positive control system exposed to bacterial endotoxin (120 ng/mℓ) were 153.5 ±17 and 1 561 ±30 pg/mℓ, respectively (p= 0.03).The production of IL-6 was unaffected following exposure of MNL to the control water samples.However, inlet water sampled from all three facilities, especially Hartbeespoort, resulted in significant activation of production of IL-6 by MNL, which declined with progressive treatment, consistent with removal of pro-inflammatory contaminants.Surprisingly, however, a rebound in pro-inflammatory activity was evident in outlet water sampled from Hartbeespoort.In conclusion, the results of the current study appear to support the efficiency of water treatment procedures at the Daspoort and Rietvlei Treatment Facilities, while confirming the usefulness of IL-6-based assays as adjuncts to conventional water quality testing procedures.

INTRODUCTION

Traditional water quality testing techniques test for the presence of indicator organisms (Hunter, 2003).However, certain substances such as endotoxin may still be present in the water, and are rarely detected by traditional methods (Wichmann et al., 2004;Gorbet and Sefton, 2005;Slabbert et al., 2008;Pool, 2008).The inflammatory activity in environmental water samples has been investigated using in vitro human immune response tests, specifically the production of pro-inflammatory cytokines by whole blood or isolated leukocytes (Pool et al., 2003;Wichmann et al., 2004;Pool and Magcwebeba, 2009).In the study reported by Wichmann et al. (2004), exposure to treated drinking water samples did not induce cytokine secretion by human leukocytes in vitro, while contrasting effects were observed following exposure to all the river water samples tested, confirming the findings of an earlier study (Pool et al., 2003).These latter authors reported that induction of secretion of the cytokine, interleukin (IL)-6, was dependent on the concentration of the contaminant(s) and origin of the water samples.More recently, exposure of whole blood from healthy adult human volunteers to hydrophobic extracts of contaminated surface water samples resulted in the synthesis of pro-inflammatory IL-6, while suppressing biomarkers of both cell-mediated and humoral immunity (Pool and Magcwebeba, 2009).In addition, several reports have documented that treated drinking water samples, sewage effluents entering river water consumed by humans, and river water treated with insecticides, possess pro-inflammatory activities (Pool, 2008;Slabbert et al., 2008;Mompelat et al., 2009).Clearly, difficultto-detect pro-inflammatory substances in water may pose a threat to public health.

With increasing awareness of this threat, the measurement of the pro-inflammatory activity of drinking water from various sources is clearly a useful adjunctive strategy to complement traditional testing procedures, as described in several previous reports alluded to above (Pool et al., 2003;Poole et al., 2003;Wichmann et al., 2004;Pool and Magcwebeba, 2009).Although these procedures have been used to assess the human health-related quality of water from various sources in several provinces in South Africa, they have not, to our knowledge, been used in the comparative analysis of water from major treatment facilities in Gauteng Province.Accordingly, the primary objective of the current study was to assess the proinflammatory activity of water samples from the Daspoort, Hartbeespoort, and Rietvlei treatment facilities, using

METHODS

Preparation of mononuclear leukocytes (MNL)

Permission to draw blood from healthy, non-smoking adult human volunteers was granted by the Research Ethics Committee of the Faculty of Health Sciences of the University of Pretoria (Protocol number 43/2006, date of renewal: 29 June 2009).Subsequent to a satisfactory health check by a qualified nursing sister and obtaining informed consent, MNL were prepared from heparinised (5 units of preservative-free heparin/ mℓ) venous blood from 3 different donors.Following centrifugation of heparinised whole blood on Histopaque-1077 cushions (Sigma-Aldrich, St Louis, MO, USA), the MNL fraction at the plasma/Histopaque-1077 interface was aspirated, diluted 1/5 with phosphate-buffered saline (PBS, 0.15 M, pH 7.4) and the cells pelleted by centrifugation, after which contaminating erythrocytes were removed by hypotonic lysis with 0.83% ammonium chloride.Subsequent to another centrifugation step and discarding of the supernatant fluid, the cells were re-suspended in sterile tissue culture medium RPMI 1640 (Highveld Biological, Johannesburg) containing antibiotics (penicillin: streptomycin: amphotericin B, 0.1: 0.25: 0.1 µg/mℓ).The MNL preparation was then analysed flow cytometrically using a Beckman Coulter FC500 Flow Cytometer using the following fluorochrome-labelled monoclonal antibodies (Beckman Coulter, Miami, FL, USA): CD3 (FITC), CD15 (FITC), CD14 (PE), and CD19 (PE) for analysis and enumeration of total T cells, neutrophils, monocytes and B-cells, respectively.The cell preparations were adjusted to a concentration of 1x10 7 /mℓ in RPMI 1640 with antibiotics and viability determined flow cytometrically using a propidium iodide dye (50 µg/mℓ, final) exclusion procedure.The cells (2x10 5 /mℓ) were incubated with the dye for 5 min at 37°C, analysed flow cytometrically and the results expressed as % viable cells.

Collection of water samples

Water samples (10 mℓ) were collected between February and April 2010, into sterile universal containers, kept on ice or at 4°C during transport to the laboratory, filtered and processed within 24 h of collection.For the assay of pro-inflammatory activity, water samples were filtered using Whatman filters of pore size 0.22 μm.Control water samples were collected from: (i) domestic taps (2 sources in Johannesburg and Pretoria); (ii) a commercial source (bottled water); and (iii) distilled water, all of which were presumed to be well treated and potable for domestic and drinking purposes.Environmental water samples were taken from 3 different water treatment facilities in Pretoria and Hartbeespoort, South Africa.The selected facilities included the Hartbeespoort Dam Water Treatment Plant (GPS: 25° 43' 530" S, 27° 50' 873" E), the Daspoort Sewage Treatment Plant (GPS: 25° 43' 16.72" S, 28° 03' 14.07" E) and the Rietvlei Dam Water Treatment Facility (GPS: 25° 52' 34" S, 28° 15' 54" E).Samples were collected at various stages of treatment, from the entry of untreated raw water (inlet water), through the final stages of treatment (outlet), and also at domestic taps supplied by the treatment plants.All samples were treated identically using RPMI 1640 with antibiotics as the diluent.

Interleukin-6 (IL-6) assay MNL (1 x 10 6 ) suspended in 900 μℓ RPMI 1640 supplemented with 5% autologous serum and antibiotics in 5 mℓ tissue culture tubes were pre-incubated for 15 min at 37°C followed by addition of 100 μℓ of one of the following to the various control and experimental systems: (i) RPMI 1640 containing antibiotics (negative control system); (ii) bacterial lipopolysaccharide, (Sigma-Aldrich), an activator of IL-6 synthesis, at a final, fixed concentration of 120 ng/mℓ (positive control system); and (iii) the test water samples, either undiluted, or diluted with RPMI 1640 containing antibiotics.The final volume in each tube was 1 mℓ, and the tubes were then incubated for 18 h at 37°C in an atmosphere of 5% CO 2 .Following incubation, the MNL were pelleted by centrifuging the tubes at 1 200 r/min for 10 min and the cell-free supernatants aspirated into sterile Eppendorf tubes and assayed for extracellular IL-6 using a commercial quantitative sandwich enzyme immunoassay procedure according to the manufacturer's instructions (Quantikine® Human IL-6, R&D Systems Inc., MN, USA) and the results expressed as pg/mℓ supernatant.

Expression and statistical analysis of results

A total of 3 different experiments using MNL from 3 different donors were performed with triplicate determinations for each system in each experiment.The results are expressed as the mean values ± standard deviation (SD), while statistical analyses were performed using GraphPad Instat version 3. The probability value (p) was calculated using a non-parametric unpaired, two-tailed Mann-Whitney test.

RESULTS

Cellular composition of MNL suspensions

The mean numbers (± SDs) of leukocyte subsets in the MNL suspensions from the 3 different donors were as follows: 54.4 ± 9.1% T cells, 15.7 ± 6.3% B cells, 10.8 ± 6.6% monocytes and 2.1 ± 1.3% contaminating neutrophils.The balance is likely to consist of natural killer cells and other leukocyte subtypes represented at low concentrations in blood.The mean percentage MNL viability was 97.5 ± 0.4% (n=3).

Effect of exposure of MNL to control water samples on IL-6 production

These results are shown in Fig. 1 which, relative to the negative control system, demonstrates a clear lack of effect of exposure of MNL to the various control water samples on IL-6 production.In the case of the positive control system, exposure of the cells to bacterial endotoxin resulted in a statistically significant (p = 0.03), substantial increase in synthesis of IL-6.

Effect of exposure of MNL to environmental water samples on IL-6 production

These results, which are shown in Fig. 2, demonstrate that several of the water samples tested induced statistically significant (p < 0.0001) induction of synthesis of IL-6 by exposed MNL compared with the negative control systems, though the degree to which this occured varied according to the origins of the samples.Those which were most potent with respect to induction of IL-6 synthesis originated from the inlet source at the

381

Hartbeespoort Dam Water Treatment Facility, reaching a maximum value of 1 094 pg/mℓ, considerably higher than the value of the corresponding negative control system.Although lower than the Hartbeespoort value, the inlet water samples from the Daspoort treatment facility also induced significant induction of IL-6 (p = 0.0001) when added to MNL.In all cases, successive treatments resulted in progressive loss of pro-inflammatory activity, which in the case of the Daspoort and Rietvlei facilities was essentially complete at the post/outlet/storage points.Surprisingly, however, despite statistically significant decreases at the pre/post treatment phase, a rebound in pro-inflammatory activity was detected in outlet water from the Hartbeespoort Treatment Facility (p < 0.0001).

Effects of dilution on the pro-inflammatory activity of inlet and outlet water from the Hartbeespoort Dam Water Treatment Plant

Inlet water samples from the Hartbeespoort Dam Water Facility were very turbid.Accordingly, dilutions of 1/10 and 1/100 in RPMI 1640 with antibiotics were made of both inlet and outlet water samples after filtration to assess potency with respect to induction of IL-6 production by MNL.These results, which are shown in Fig. 3, demonstrate no loss of pro-inflammatory potency of inlet water following dilution of 1/10 (p = 0.9045), while there is clear loss of pro-inflammatory potency of outlet water at a dilution of 1/10 (p = 0.0007).

Effects of exposure of MNLs to: (i) RPMI 1640 with antibiotics (negative control); (ii) bacterial endotoxin (120 ng/mℓ, positive control); (iii) DT1 (domestic tap water, Johannesburg); (iv) DT2 (domestic tap water, Pretoria); (v) BW (commercially bottled water); (vi) DW (distilled water); and (vii) LW (laboratory tap water). The results of 3 separate experiments using MNL from 3 different donors are expressed as the mean values ±

SDs with triplicate determinations for each system in each experiment.*The value for the positive control system was significantly higher (p<0.03)than those for the negative control system and the various water sources, which did not differ significantly from one another.

Figure 2 (left)

Comparison of the effects of water sampled from the 3 different treatment facilities, before, during and after treatment, on the production of IL-6 by isolated human MNLs.The results of 3 separate experiments using MNL from 3 different donors are expressed as the mean values ±SDs with triplicate determinations for each system in each experiment.In all cases, the pre-treatment, post-treatment, outlet and storage values were significantly lower ( * p<0.0001) than those of the corresponding inlet values for each water treatment facility.

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DISCUSSION

Various water contaminants, including bacteria, viruses and their breakdown products, have been shown to cause adverse health effects in humans, which are characterised by fever, diarrhoea, and, in extreme cases, anaphylactic shock (Emmanuel et al., 2007).Traditional water treatment procedures such as chlorination are designed to eliminate living organisms, but chlorination has little or no effects on bacterial breakdown products such as endotoxin (Gorbet and Sefton, 2005).Although some assays such as the limulus amoebocyte lysate (LAL) test detect microbial breakdown products such as Gram-negative bacterial endotoxin, which may be present in river and drinking water (Poole et al., 2003;Wichmann et al., 2004;Gorbet and Sefton, 2005;Pool, 2008), these procedures are ineffective in the detection of other pyrogenic substances (Gorbet and Sefton, 2005;Hodgson, 2006).

Alternative assays include assessment of the inflammatory activity of water as an indirect index of quality.In this setting, measurement of the cytokine IL-6, following exposure of whole blood or isolated leukocytes to test water samples in vitro, is a useful strategy to detect inflammatory activity (Wichmann et al., 2004;Pool and Magcwebeba, 2009).This procedure was used in the current study to determine the quality of water sampled at various stages prior to, during, and following the treatment process at 3 water treatment facilities in the greater Pretoria region, relative to that of control water samples.

The results revealed that none of the control water samples tested affected the production of IL-6 by MNL isolated from the blood of 3 healthy adult human volunteers, the levels of production of the cytokine being comparable with those of the negative control systems.In the case of the water treatment facilities (Daspoort, Hartbeespoort and Rietvlei), however, inlet water from all three, especially Hartbeespoort, was found, not unexpectedly, to possess significant inflammatory activity.The purification procedures in all three treatment facilities were associated with a progressive loss of pro-inflammatory activity, with the values for outlet and storage water sampled from the Daspoort and Rietvlei facilities being equivalent to or lower than that of the negative control system.

Surprisingly, however, a rebound in pro-inflammatory activity was detected in the outlet water, in particular, and, to a lesser extent, the storage water, sampled from the Hartbeespoort facility.We are not aware of previous studies which have investigated the inflammatory potential of Hartbeespoort Dam water.Although the possible health hazards of consumption of water with residual inflammatory activity are unknown, our findings are consistent with reports of poor water quality, with users often complaining of the unpleasant odour of treated water which originated from the Hartbeespoort Dam Water Treatment Facility (Dower, 2005;Venter, 2005;DWAF, 2008).Hartbeespoort Dam is well-known for its algae blooms (blue-green algae), especially cyanobacteria species and their microcystin toxins, as well as other bacteria known to produce toxins (Venter, 2005;DWAF, 2008).Although many of these toxins are heat-resistant and not affected by filtration and chlorination (Gorbet and Sefton, 2005), the progressive loss of inflammatory activity appears to exclude inefficiency of the treatment processes as the reason for the unexpectedly high level of inflammatory activity in the outlet and storage water.Alternative possibilities include equipment failures, re-contamination, improper disinfection processes, and inadequate removal of disinfectants, poor storage practices, inadequate or unskilled manpower or a combination of these factors, which might have contributed to the inflammatory reactivity of the outlet water at the time of sampling.Determining the actual factors responsible for the inflammatory reactivity in the water samples was, however, outside the scope of the present study.

In the case of the Daspoort Sewage Treatment Facility, the inlet water consists of sewage and waste water effluents from Pretoria, presumably rich in biological wastes with a high concentration of bacteria, e.g., coliforms and E. coli.This probably explains the relatively high inflammatory reactivity seen at the inlet point, which decreased progressively as the water passed through the treatment processes.The operating principles of the Daspoort Waste Water Treatment Works, incorporating the external nitrification biological nutrient removal activated sludge system for increased efficiency, have been described in detail by Muller et al. (2004).The treated water pumped to the hydro-powered station and back into the Apies River (storage water for Daspoort Dam Water Treatment Plant) was not potentially harmful, which is in agreement with the report by Muller et al. (2004), and is important for the maintenance of biodiversity in the aquatic environment.The Apies River water flows down to the Delmas Water Treatment Facility to be treated for domestic use.

Although the Rietvlei Dam flows through industrial and residential areas (City of Tshwane, 2003), the results showed that it is the least polluted water of the environmental water samples tested in this study.This may be attributed to the fact that water flowing into the dam is derived from 4 natural springs and very few effluents are discharged into the dam (City of Tshwane, 2003).Although occasional filter blockages have been reported at the Rietvlei Dam Water Treatment Plant due to abnormal algal blooms in the upper layer of Rietvlei Dam, especially during winter (Rietvlei Dam, 2009), reports of poor water quality are very rare.These algae species are effectively removed from the water by pre-chlorination and backwashing at more regular intervals (City of Tshwane, 2009).In addition, a wetland occurs upstream of the Rietvlei Dam which slows down water flow, causing suspended matter to settle out.The Rietvlei Dam Water Treatment Plant supplies 5% of the domestic and industrial water needs of the Tshwane Municipality, and reports of poor water quality or taste are rare (City of Tshwane, 2003).

We do concede that the current study suffers from several limitations, including the relatively small number of blood donors (n=3) and lack of serial sampling over an extended period.Nevertheless, the results of this pilot study appear to identify varying efficiencies of the water treatment practices at three major treatment facilities in the greater Pretoria region, while confirming the usefulness of testing for inflammatory potential as an adjunct to traditional water testing procedures.

Figure 3
3
Figure 3Effects of dilution ( 1 / 10 and/or 1 / 100 ) of inlet and outlet water sampled from the Hartbeespoort Water Treatment Facility on the production of IL-6 by isolated human MNLs.The results of 3 experiments using cells from 3 different donors are expressed as the mean values ±SDs with triplicate determinations for each system in each experiment.The positive and negative control systems contained bacterial endotoxin (120 ng/mℓ) and an equivalent volume of RPMI 1640 containing antibiotics, respectively; *p<0.0001 for comparison with the corresponding undiluted systems.

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