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Measuring Artificial Sweeteners Toxicity Using a Bioluminescent Bacterial Panel

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Artificial sweeteners have become increasingly controversial due to their questionable influence on consumers’ health. They are introduced in most foods and many consume this added ingredient without their knowledge. Currently, there is still no consensus regarding the health consequences of artificial sweeteners intake as they have not been fully investigated. Consumption of artificial sweeteners has been linked with adverse effects such as cancer, weight gain, metabolic disorders, type-2 diabetes and alteration of gut microbiota activity. Moreover, artificial sweeteners have been identified as emerging environmental pollutants, and can be found in receiving waters, i.e., surface waters, groundwater aquifers and drinking waters. In this study, the relative toxicity of six FDA-approved artificial sweeteners (aspartame, sucralose, saccharine, neotame, advantame and acesulfame potassium-k (ace-k)) and that of ten sport supplements containing these artificial sweeteners, were tested using genetically modified bioluminescent bacteria from E. coli. The bioluminescent bacteria, which luminesce when they detect toxicants, act as a sensing model representative of the complex microbial system. Both induced luminescent signals and bacterial growth were measured. Toxic effects were found when the bacteria were exposed to certain concentrations of the artificial sweeteners. In the bioluminescence activity assay, two toxicity response patterns were observed, namely, the induction and inhibition of the bioluminescent signal. An inhibition response pattern may be observed in the response of sucralose in all the tested strains: TV1061 (MLIC = 1 mg/mL), DPD2544 (MLIC = 50 mg/mL) and DPD2794 (MLIC = 100 mg/mL). It is also observed in neotame in the DPD2544 (MLIC = 2 mg/mL) strain. On the other hand, the induction response pattern may be observed in its response in saccharin in TV1061 (MLIndC = 5 mg/mL) and DPD2794 (MLIndC = 5 mg/mL) strains, aspartame in DPD2794 (MLIndC = 4 mg/mL) strain, and ace-k in DPD2794 (MLIndC = 10 mg/mL) strain. The results of this study may help in understanding the relative toxicity of artificial sweeteners on E. coli, a sensing model representative of the gut bacteria. Furthermore, the tested bioluminescent bacterial panel can potentially be used for detecting artificial sweeteners in the environment, using a specific mode-of-action pattern.
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molecules
Article
Measuring Artificial Sweeteners Toxicity Using a
Bioluminescent Bacterial Panel
Dorin Harpaz 1,2,3 , Loo Pin Yeo 1, Francesca Cecchini 4, Trish H. P. Koon 5,6,
Ariel Kushmaro 2,7,8, Alfred I. Y. Tok 1,3, Robert S. Marks 2,7,8,* and Evgeni Eltzov 9, *
1School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue,
Singapore 639798, Singapore; DORIN001@e.ntu.edu.sg (D.H.); yeolp@ntu.edu.sg (L.P.Y.);
MIYTok@ntu.edu.sg (A.I.Y.T.)
2Avram and Stella Goldstein-Goren, Department of Biotechnology Engineering, Faculty of Engineering
Sciences, Ben Gurion University of the Negev, Beer-Sheva 84105, Israel; arielkus@bgu.ac.il
3Institute for Sports Research (ISR), Nanyang Technology University and Loughborough University,
Nanyang Avenue, Singapore 639798, Singapore
4TURVAL Laboratories, Ltd. (Laboratori Turval Italia Srl), via J. Linussio 51, 33100 Udine, Italy;
cecchini@turval.com
5Department of Obstetrics and Gynaecology, KK Women’s and Children’s Hospital, 100 Bukit Timah Road,
Singapore 229899, Singapore; trishkoon@gmail.com
6School of Science and Technology, Singapore University of Social Sciences, 463 Clementi Road,
Singapore 599494, Singapore
7The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev,
Beer-Sheva 84105, Israel
8
The Ilse Katz Centre for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev,
Beer-Sheva 84105, Israel
9Agriculture Research Organization (ARO), Volcani Centre, Rishon LeTsiyon 15159, Israel
*Correspondence: rsmarks@bgu.ac.il (R.S.M.); eltzov@volcani.agri.gov.il (E.E.)
Received: 6 August 2018; Accepted: 22 September 2018; Published: 25 September 2018


Abstract:
Artificial sweeteners have become increasingly controversial due to their questionable
influence on consumers’ health. They are introduced in most foods and many consume this
added ingredient without their knowledge. Currently, there is still no consensus regarding
the health consequences of artificial sweeteners intake as they have not been fully investigated.
Consumption of artificial sweeteners has been linked with adverse effects such as cancer, weight gain,
metabolic disorders, type-2 diabetes and alteration of gut microbiota activity. Moreover, artificial
sweeteners have been identified as emerging environmental pollutants, and can be found in receiving
waters, i.e., surface waters, groundwater aquifers and drinking waters. In this study, the relative
toxicity of six FDA-approved artificial sweeteners (aspartame, sucralose, saccharine, neotame,
advantame and acesulfame potassium-k (ace-k)) and that of ten sport supplements containing
these artificial sweeteners, were tested using genetically modified bioluminescent bacteria from
E. coli. The bioluminescent bacteria, which luminesce when they detect toxicants, act as a sensing
model representative of the complex microbial system. Both induced luminescent signals and
bacterial growth were measured. Toxic effects were found when the bacteria were exposed to
certain concentrations of the artificial sweeteners. In the bioluminescence activity assay, two toxicity
response patterns were observed, namely, the induction and inhibition of the bioluminescent signal.
An inhibition response pattern may be observed in the response of sucralose in all the tested strains:
TV1061 (
MLIC = 1 mg/mL
), DPD2544 (MLIC = 50 mg/mL) and DPD2794 (
MLIC = 100 mg/mL
). It is
also observed in neotame in the DPD2544 (MLIC = 2 mg/mL) strain. On the other hand, the induction
response pattern may be observed in its response in saccharin in TV1061 (
MLIndC = 5 mg/mL
) and
DPD2794 (MLIndC = 5 mg/mL) strains, aspartame in DPD2794 (MLIndC = 4 mg/mL) strain,
and ace-k in DPD2794 (MLIndC = 10 mg/mL) strain. The results of this study may help in
understanding the relative toxicity of artificial sweeteners on E. coli, a sensing model representative
Molecules 2018,23, 2454; doi:10.3390/molecules23102454 www.mdpi.com/journal/molecules
Molecules 2018,23, 2454 2 of 20
of the gut bacteria. Furthermore, the tested bioluminescent bacterial panel can potentially be used for
detecting artificial sweeteners in the environment, using a specific mode-of-action pattern.
Keywords:
artificial sweeteners; sport supplements; bioluminescent bacteria; toxic effect;
gut microbiota; environmental pollutants
1. Introduction
Artificial sweeteners are an important class of sugar substitutes known as high-intensity
sweeteners (HIS), also referred to as non-nutritive sweeteners (NSS) or as non-caloric sweeteners
(NCS) [
1
]. The Food and Drug Authority (FDA) has approved the use of six artificial sweeteners,
which includes aspartame, sucralose, saccharin, advantame, neotame and acesulfame potassium-k
(ace-k), in food and beverages [
2
]. The recent EU legislation has also approved of these artificial
sweeteners [
3
]. Artificial sweeteners provide a sweeter taste than sugar and also enhance food flavor,
while contributing very little to energy intake [
4
]. These sweeteners are most commonly used as food
additives [
5
]. Many different population groups consume the added ingredient, with or without their
knowledge. This is especially common with athletes who devote full-time care to their diet, which often
include sport supplements to improve their physical performance in trainings and competitions [
6
].
In several registered products’ patents [
7
9
], it is clearly stated that artificial sweeteners are added to
electrolyte drinks and food supplements [
10
14
]. As a result, the average consumption of artificial
sweeteners is higher in athletes and any potential health risks involved would also be more significant.
The health risks of artificial sweeteners consumption is still a highly controversial topic [
15
].
Artificial sweeteners have allegedly been linked to adverse effects such as cancer, weight gain, metabolic
disorders, migraines, type-2 diabetes, vascular events, preterm delivery, kidney function disorders,
liver antioxidant system, hepatotoxicity, immune system disruptions and alteration of gut microbiota
activity [
16
,
17
]. Although these potential health problems have long been studied, a firm conclusion
has yet to be reached on these allegations due to a lack of consistent evidence. Subsequent human
studies failed to show a direct connection to cancer risk [
18
,
19
]. Other studies, however, have shown
association with kidney function decline [
20
] and vascular risk factors [
21
]. Consumption of artificial
sweeteners as food additives has been promoted as a prevention strategy against obesity as well as
a diet for weight loss as they replace the high-calorie sweeteners. Studies have compared a diet of
artificial sweeteners versus no artificial sweeteners and artificial sweeteners versus traditional sugars,
with results showing greater weight loss and better weight management in an artificial sweetener
diet [
22
,
23
]. However, the converse has also been proven true [
24
,
25
]. It was shown that consuming
diet soda results in more weight gain than consuming naturally-sweetened soda [
26
]. In another
study, rats given artificial sweeteners showed steadily increasing caloric intake, increased body weight,
and increased adiposity [
27
]. Since the 1980s, there have been studies reporting associations between
artificial sweeteners and alteration in bacterial composition. Subsequent studies, investigating the
possible effects of artificial sweeteners on the gut microbiota system, presented controversial results.
A recent study has shown that the intake of artificial sweeteners such as lactitol or maltitol increased
some beneficial bacteria such as lactobacillus in the gut system [
28
]. A second study concluded that
artificial sweeteners induce glucose intolerance. Mice were fed with artificial sweeteners in drinking
water and demonstrated gut microbiota changes [
29
]. Another related study was conducted on pigs fed
with artificial sweeteners but it was concluded that there is a selective effect on the gut microbiota [
30
].
Moreover, artificial sweeteners have been identified as emerging environmental pollutants [
31
,
32
].
They are resistant to wastewater treatment processes, therefore they are continuously introduced
into the water environments [
33
]. Several environmental studies have confirmed the widespread
distribution of ace-k, saccharin and sucralose in the water cycle [
34
39
]. Concentrations of ace-k and
sucralose up to the
µ
g L
1
range can be found in receiving waters, i.e., surface waters, groundwater
Molecules 2018,23, 2454 3 of 20
aquifers and drinking waters. Such concentrations are among the highest known for anthropogenic
trace pollutants [31,40].
Typically, the toxicological evidence is derived from studies in appropriate animal models,
and possibly from human trials. The compounds can be evaluated across a wide range of exposures,
including duration and persistence of exposures [
41
]. Other associated manifestations of toxicity in
humans may be identified from the case reports and epidemiological studies after product marketing.
However, all of these approaches are time-consuming and expensive. Thus, there is a demand
for fast and simple approaches that will provide toxicity evaluation of the artificial sweeteners.
Progress in the genetic engineering field allows not only the “tailoring” of microorganisms for
determining the identity of the target analyte but also allows the monitoring of the biological
activity of these chemicals by analyzing different cell responses (e.g., gene expression, metabolic
activity, viability). In this study, bacteria engineered to luminesce after exposure to certain stresses
were used [
42
]. The bioluminescent bacteria, which luminesce when they detect toxicants, act as
a sensing model representative of the complex microbial system. The relative toxicity of the six
FDA approved pure artificial sweeteners (aspartame, sucralose, saccharine, neotame, advantame and
ace-k) and 10 sport supplements containing artificial sweeteners were tested using three different
E. coli strains (TV1061, DPD2544 and DPD2794) of genetically modified bioluminescent bacteria
sensitive to the various stresses (e.g., cytotoxicity, amino acids availability, genotoxicity, accordingly).
Luminescence was easily measured using a sensitive photodetector unaffected by variable background
signals [
43
]. The bioreporter bacteria developed in this study used luciferase as the reporter gene,
which provided a sensitive and simple detection method for gene expression and regulation [
44
].
An additional advantage of a bacterial luciferase-based bioassay is the ability to express a whole
luciferase operon that produces a luminescent cell without any additions or external existing sources,
thus allowing for real-time monitoring of gene expression [
42
]. Expressed effectively in different strains,
the bacterial lux system has been used for sensing various compounds such as heavy metals [
45
47
],
androgen-like [
48
,
49
], active oxygen [
50
], endocrine disrupting chemicals [
51
], phenolics [
52
] and other
environmental pollutants [
53
57
]. The simplicity and biological relevance of bacterial bioreporter
assays makes them attractive as a rapid and cheap monitoring method of the presence of toxicants in
water, air, soil and food samples [58].
2. Results
2.1. Artificial Sweeteners Toxicity and Viability Effect
The biological effect of the artificial sweeteners on the bioluminescent bacteria was quantified
using a toxicity index. This index describes the response ratios between treated and untreated
microorganisms and may provide information about the possible toxicity of the artificial sweeteners.
Figure 1shows three different response patterns of the bioreporter bacteria to the tested chemicals.
The first toxicity response pattern is signal induction, where increased chemical concentration
induced bacterial luminescence. This may be observed in the response of the TV1061 strain to
saccharin (Figure 1A) and DPD2794 strain to aspartame and saccharin (Figure 1C). In all of these
cases, dose dependent responses were observed, where greater chemical concentrations produced
higher inducing effect. The second toxicity response pattern is inhibition, where increased chemical
concentrations decreased cells luminescence (response of the TV1061 strain to sucralose (Figure 1A)).
As in the previous case, in an inhibition pattern, higher concentrations have a stronger biological
effect. For the third response pattern, no visible effect was observed through all tested concentrations
(Figure 1B).
Molecules 2018,23, 2454 4 of 20
Molecules 2018, 23, x FOR PEER REVIEW 4 of 20
Figure 1. Artificial sweeteners toxicity. The toxicity index of different artificial sweeteners on the three
tested bioluminescent bacteria strains: (A) TV1061; (B) DPD2544; (C) DPD2794. A strong induction
response pattern may be observed in the response of the TV1061 strain to saccharin and DPD2794
strain to aspartame and saccharin. In addition, a strong inhibition response pattern may be observed
in the response of the TV1061 strain to sucralose.
Figure 1.
Artificial sweeteners toxicity. The toxicity index of different artificial sweeteners on the three
tested bioluminescent bacteria strains: (
A
) TV1061; (
B
) DPD2544; (
C
) DPD2794. A strong induction
response pattern may be observed in the response of the TV1061 strain to saccharin and DPD2794
strain to aspartame and saccharin. In addition, a strong inhibition response pattern may be observed in
the response of the TV1061 strain to sucralose.
Molecules 2018,23, 2454 5 of 20
During this study, we exposed different bioluminescent bacterial strains to various artificial
sweeteners (Table 1), and compared their minimum luminescent inhibition concentration (MLIC),
minimum luminescence induction concentration (MLIndC), minimum growth inhibition concentration
(MGIC) and minimum growth induction concentration (MGIndC). The logic behind this method is
that, during luminescent activation, the promoters fuse to the lux reporter genes and will not only show
the possible toxic effects of the artificial sweeteners but will also create a specific pattern. This allows
us to infer the mode of action of the sport supplements through the induced bioluminescence pattern
of specific bioreporter bacteria. From all the tested artificial sweeteners, only sucralose and neotame
inhibited bioluminescent responses of the bioreporter bacteria (Table 1). Neotame reduces light
response only in the DPD2544 strain while sucralose inhibits it in all the tested strains. Furthermore,
in sucralose, this inhibition effect was observed not only with luminescence but also with bacterial
growth (Figure S1 presented in Supplmentary Data). While the cells’ growth rates were affected by the
same MGIC of 50 mg/mL in all strains, the luminescent signals were affected by different sweetener
concentrations depending on the strains used. The light response of the DPD2794, SOS-dependent
bioluminescence strain was inhibited by a sucralose concentration that was two-fold higher than that
needed for the TV1061 cytotoxic strain. Interestingly, an induction effect was observed only for the
cases of TV1061 with saccharin and neotame, and DPD2794 with aspartame, saccharin and ace-k.
Table 1. Artificial sweeteners toxicity and viability effect (mg/mL).
Strain MLIC MLIndC MGIC MGIndC
Aspartame
TV1061 N.E. N.E. N.E. N.E.
DPD2544 N.E. N.E. N.E. N.E.
DPD2794 N.E. 4 N.E. N.E.
Sucralose
TV1061 1 N.E. 50 N.E.
DPD2544 50 N.E. 50 N.E.
DPD2794 100 N.E. 50 N.E.
Saccharin
TV1061 N.E. 5 5 N.E.
DPD2544 N.E. N.E. N.E. N.E.
DPD2794 N.E. 5 N.E. N.E.
Advantame
TV1061 N.E. N.E. N.E. 2
DPD2544 N.E. N.E. N.E. N.E.
DPD2794 N.E. N.E. N.E. N.E.
Neotame
TV1061 N.E. 2 N.E. N.E.
DPD2544 2 N.E. N.E. N.E.
DPD2794 N.E. N.E. N.E. N.E.
Ace-K
TV1061 N.E. N.E. N.E. N.E.
DPD2544 N.E. N.E. N.E. N.E.
DPD2794 N.E. 10 N.E. N.E.
MLIC—Minimum Luminescent Inhibition Concentration; MLIndC—Minimum Luminescent Induction
Concentration; MGIC—Minimum Growth Inhibition Concentration; MGIndC—Minimum Growth Induction
Concentration; N.E.—No Effect.
In general, from all tested strains, TV1061 was the most susceptible to artificial sweeteners.
The lowest inhibition and induction concentrations that resulted in a toxic response were observed with
sucralose (1 mg/mL) and neotame (2 mg/mL), respectively. A light induction effect was observed only
within TV1061 and DPD2794 strains. Furthermore, 5 mg/mL saccharin induced TV1061 luminescence
while also showing a growth inhibition effect. In general, several induction and growth patterns may be
observed, e.g., luminescence induction (DPD2794 with aspartame, saccharin, ace-K, and TV1061 with
neotame), growth induction (TV1061 with advantame), and the combination of luminescence induction
with growth inhibition (TV1061 with saccharin). From all the tested additives, only advantame induced
growth without any visible effect on the luminescence signal. For the other tested chemicals, the MGIC
parameter could not be estimated, with the maximum achievable concentration not showing any
Molecules 2018,23, 2454 6 of 20
visible effect in all tested strains. To conclude, for all tested artificial sweeteners, the luminescence and
growth effect pattern were either fully inhibition or induction, without any case of a combination of
both toxicity response patterns within the same sample.
2.2. Sport Supplements Toxicity and Viability Effect
The response patterns of the bioluminescent bacteria strains to the chemicals present in sport
supplements were also tested and the results are presented in Table 2. Different response patterns were
obtained for each tested strain. The response pattern of the bacteria strains to the sports supplements
were contrary to their response to the artificial sweeteners. The DPD2544 luminescence signal was
inhibited by all of the sport supplements. Simultaneously, the luminescence signal of this strain also
showed induction response albeit at lower concentrations of the sport supplement. In all samples,
the MLIndC values (pg/mL) were 1000-fold lower than the MLIC values (ng/mL). Nevertheless,
the sport supplements did not have any effect on the DPD2544 growth rate (Table 2). Conversely,
the other two bacteria strains were inhibited only when exposed to a specific sport supplement,
DPD2794 with SS3 and TV1061 with SS7.
The bacterial strains responded differently to the varying sport supplements, with four response
patterns observed. In the first response pattern, only cell bioluminescence (MLIndC) was induced.
In this case, MGIC or MGIndC parameters cannot be estimated as the maximum achievable
concentration did not have any visible effect. MLIndC, however, was quantitative. Such response
pattern was observed when the TV1061 strain was exposed to SS1, SS5, SS7 and SS9 sport supplements.
In the second response pattern, in addition to the bioluminescence induction or inhibition, the bacteria
growth rates were also affected (Figure S2 presented in Supplmentary Data). This is observed in the
response of the DPD2794 to SS3 (MLIndC with MGIC) or SS5 (MLIC with MGIndC) (Table 2). In the
third response pattern, only the growth rate of the bioreporter bacteria was affected by the sport
supplements. While the supplements either inhibited (SS1, SS2, SS5, SS7) or induced (SS3, SS4, SS6,
SS8, SS10) the growth rates of TV1061 and DPD2794 strains, only SS9 did not show any visible effect
on the bacteria growth rate. In the last pattern, more than two trends in the bacterial responses were
observed. For example, when SS7 was exposed to TV1061, it not only induced bacterial growth rates
but also increased and decreased (at different concentrations) the cells bioluminescence.
The changes in the cell bioluminescence when exposed to the different concentrations of the
three sport supplements (SS3, SS5, and SS7) are presented in Figure 2. The response of strain TV1061
(with heat-shock gene grpE fused to lux gene) to different sport additives (Figure 2A) resembles
its response pattern to the artificial sweeteners (Figure 1A), whereby increasing concentrations of
the sport supplements in the tested sample increased its biological effect on the TV061 strains in a
dose-dependent manner. For example, at higher SS3 concentrations, the cell luminescence increased,
whereas the SS7 showed increasing inhibition effect. The response of strain DPD2544 is also very
similar to the cells’ reaction to the artificial sweeteners (Figure 2B). The main difference is that, at lower
concentrations of sport supplements, the bacterial luminescence signals are induced, while, at higher
concentrations, they are inhibitory. The DPD2794 also responded in a dose-dependent manner to the
sport supplements, whereby an induction response in bioluminescence was observed in SS5 and an
inhibition response was observed in SS3 and SS7. In general, at lower concentrations, the intensity of
the signal received in response to the sport supplements was slightly lower than those produced by the
exposure to the artificial sweeteners. However, at higher sample concentrations, the sport supplements
showed a stronger induction or inhibition effect.
Molecules 2018,23, 2454 7 of 20
Table 2. Sport supplements’ toxicity and viability effect (µg/mL).
Strain MLIC MLIndC MGIC MGIndC
SS1
TV1061 N.E. 2000 N.E. N.E.
DPD2544 2×1032×106N.E. N.E.
DPD2794 N.E. N.E. 2000 N.E.
SS2
TV1061 N.E. N.E. N.E. N.E.
DPD2544 1×1031×106N.E. N.E.
DPD2794 N.E. N.E. 1000 N.E.
SS3
TV1061 N.E. N.E. N.E. 4000
DPD2544 4×1034×106N.E. N.E.
DPD2794 4000 N.E. N.E. 4000
SS4
TV1061 N.E. N.E. N.E. 5000
DPD2544 5×1035×106N.E. N.E.
DPD2794 N.E. N.E. N.E. 5000
SS5
TV1061 N.E. 5000 N.E. N.E.
DPD2544 5×1035×106N.E. N.E.
DPD2794 N.E. 5000 5000 N.E.
SS6
TV1061 N.E. N.E. N.E. 3000
DPD2544 3×1033×106N.E. N.E.
DPD2794 N.E. N.E. N.E. N.E.
SS7
TV1061 5000 500 5000 N.E.
DPD2544 5×1035×106N.E. N.E.
DPD2794 5000 N.E. 5000 N.E.
SS8
TV1061 N.E. N.E. N.E. 2000
DPD2544 2×1032×106N.E. N.E.
DPD2794 N.E. N.E. N.E. 2000
SS9
TV1061 N.E. 3000 N.E. N.E.
DPD2544 3×1033×106N.E. N.E.
DPD2794 N.E. N.E. N.E. N.E.
SS10
TV1061 N.E. N.E. N.E. 3000
DPD2544 3×1033×106N.E. N.E.
DPD2794 N.E. N.E. N.E. 3000
MLIC—Minimum Luminescent Inhibition Concentration; MLIndC—Minimum Luminescent Induction
Concentration; MGIC—Minimum Growth Inhibition Concentration; MGIndC—Minimum Growth Induction
Concentration; N.E.—No Effect.
Molecules 2018, 23, x FOR PEER REVIEW 7 of 20
Table 2. Sport supplements toxicity and viability effect (µg/mL).
MLIC
MLIndC
MGIC
MGIndC
SS1
N.E.
2000
N.E.
N.E.
2 × 103
2 × 106
N.E.
N.E.
N.E.
N.E.
2000
N.E.
SS2
N.E.
N.E.
N.E.
N.E.
1 × 103
1 × 106
N.E.
N.E.
N.E.
N.E.
1000
N.E.
SS3
N.E.
N.E.
N.E.
4000
4 × 103
4 × 106
N.E.
N.E.
4000
N.E.
N.E.
4000
SS4
N.E.
N.E.
N.E.
5000
5 × 103
5 × 106
N.E.
N.E.
N.E.
N.E.
N.E.
5000
SS5
N.E.
5000
N.E.
N.E.
5 × 103
5 × 106
N.E.
N.E.
N.E.
5000
5000
N.E.
SS6
N.E.
N.E.
N.E.
3000
3 × 103
3 × 106
N.E.
N.E.
N.E.
N.E.
N.E.
N.E.
SS7
5000
500
5000
N.E.
5 × 103
5 × 106
N.E.
N.E.
5000
N.E.
5000
N.E.
SS8
N.E.
N.E.
N.E.
2000
2 × 103
2 × 106
N.E.
N.E.
N.E.
N.E.
N.E.
2000
SS9
N.E.
3000
N.E.
N.E.
3 × 103
3 × 106
N.E.
N.E.
N.E.
N.E.
N.E.
N.E.
SS10
N.E.
N.E.
N.E.
3000
3 × 103
3 × 106
N.E.
N.E.
N.E.
N.E.
N.E.
3000
MLICMinimum Luminescent Inhibition Concentration; MLIndCMinimum Luminescent
Induction Concentration; MGICMinimum Growth Inhibition Concentration; MGIndCMinimum
Growth Induction Concentration; N.E.No Effect.
Figure 2. Cont.
Molecules 2018,23, 2454 8 of 20
Molecules 2018, 23, x FOR PEER REVIEW 8 of 20
Figure 2. Sport supplements toxicity. Toxicity index of different sport supplements on the three tested
bioluminescent bacteria strains: (A) TV1061; (B) DPD2544; (C) DPD2794.
3. Discussion
3.1. Artificial Sweeteners Toxicity and Viability Effect
For decades, the food, beverages, and other industries have used artificial sweeteners as sugar
substitutes for those who are diabetic and/or obese. Industries highlight the beneficial aspects of
artificial sweeteners use, such as tooth friendliness, increased quality of life for diabetics and weight
control [59]. However, in addition to the environmental pollution issues [31], there has been much
evidence about the possible negative impact sugar substitutes contribute to human health [16
19,21,2427]. However, the total consumption of artificial sweeteners in foods has only increased
among people of all ages, with 28% of the total population consuming them [60]. For the consumers’
safety, it is necessary to control the content of sweeteners in foods. Several analytical methods
(including high-performance liquid chromatography, ion chromatography, thin-layer
chromatography, gas chromatography, capillary electrophoresis, flow-injection analysis,
electroanalysis and spectroscopy) can determine sweeteners individually and in mixtures. However,
there still remains the challenge of developing stable, reliable and robust methods for the
determination of artificial sweeteners in complex food matrices and their putative toxic effect [59].
Moreover, our diet has a direct effect on the body’s microbiome, which not only plays important
physiological roles but also reduces susceptibility to many pathophysiological conditions [61]. Thus,
the microbiome may serve as a hub, channeling the effects of one’s diet onto the hosts health and
propensity to disease. Artificial sweeteners, which are commonly found in dietary supplements, may
be subjected to the same interactions with the microbiome and thus consequently exert their effects
on the host [62]. To date, diverse methods (e.g., qPCR [63], turbidity [64], selective culturing [65],
Figure 2.
Sport supplements’ toxicity. Toxicity index of different sport supplements on the three tested
bioluminescent bacteria strains: (A) TV1061; (B) DPD2544; (C) DPD2794.
3. Discussion
3.1. Artificial Sweeteners’ Toxicity and Viability Effect
For decades, the food, beverages, and other industries have used artificial sweeteners as sugar
substitutes for those who are diabetic and/or obese. Industries highlight the beneficial aspects
of artificial sweeteners’ use, such as tooth friendliness, increased quality of life for diabetics and
weight control [
59
]. However, in addition to the environmental pollution issues [
31
], there has
been much evidence about the possible negative impact sugar substitutes contribute to human
health [
16
19
,
21
,
24
27
]. However, the total consumption of artificial sweeteners in foods has only
increased among people of all ages, with 28% of the total population consuming them [
60
]. For the
consumers’ safety, it is necessary to control the content of sweeteners in foods. Several analytical
methods (including high-performance liquid chromatography, ion chromatography, thin-layer
chromatography, gas chromatography, capillary electrophoresis, flow-injection analysis, electroanalysis
and spectroscopy) can determine sweeteners individually and in mixtures. However, there still remains
the challenge of developing stable, reliable and robust methods for the determination of artificial
sweeteners in complex food matrices and their putative toxic effect [
59
]. Moreover, our diet has
a direct effect on the body’s microbiome, which not only plays important physiological roles but
also reduces susceptibility to many pathophysiological conditions [
61
]. Thus, the microbiome may
serve as a hub, channeling the effects of one’s diet onto the host’s health and propensity to disease.
Artificial sweeteners, which are commonly found in dietary supplements, may be subjected to the same
interactions with the microbiome and thus consequently exert their effects on the host [
62
]. To date,
diverse methods (e.g., qPCR [
63
], turbidity [
64
], selective culturing [
65
], next-generation sequencing
(NGS) [
29
]) across different species have been used for the determination of potential effects of the
artificial sweeteners on microbiome. However, all such technologies are complicated, highly expensive
and time-consuming. Given this situation, there is a need for a fast and simple application for the
Molecules 2018,23, 2454 9 of 20
evaluation and characterization of the effects that artificial sweeteners (e.g., advantame, neotame,
ace-K, aspartame, saccharin, and sucralose) have on the prokaryotic cells. For example, a previous
study demonstrated the use of a whole cell microbial amperometric sensor, using an immobilized
Bacillus subtilis cells for the detection of aspartame [66].
In this study, we demonstrate the use of a panel of indicator bacteria that is able to detect active
compounds at subinhibitory concentrations and to predict the mode of action of these chemicals from
their bioluminescence responses. We use the expression of the lux gene under the control of different
stress promoters that are responsible for regulatory networks in the indicator bacteria. Three different
strains were exposed to the commercial artificial sweeteners for the determination of their possible
toxic effects (Table 1). The inhibition effect on the whole bioreporter panel was observed only with
the exposure to sucralose. Previous studies have shown that bacteria do not utilize sucralose as a
carbon source [
67
] and that the substitution of glucose with sucralose in agar medium produced a
total inhibition of growth of several strains [
68
]. In this study, sucralose induced bacterial growth at
the highest tested concentration. A possible explanation is that sucralose, which was added to the
medium containing all of the nutrients required for cell growth, did not replace the already available
carbon sources. Thus, it was not a limiting factor for the bacterial growth processes. On the other hand,
however, sucralose repressed luminescence in all the bioreporter bacteria. The MLIC values were not
only different for each strain tested (Table 1), but the strains’ kinetic responses also differed (Figure 1).
In the tested concentration range, the highest inhibition effect was observed with TV1061 strain
(1 mg/mL), and then with DPD2544 (50 mg/mL) and DPD2794 (100 mg/mL). Figure 1A demonstrates
that only the TV1061 strain showed an increasing inhibition effect with higher tested concentrations.
Such an inhibition pattern indicates that the sucralose mode of action is not affecting cyto/genotoxicity
or fatty acid synthesis pathways. Indeed, sucralose was subjected to a full battery of
in vitro
and
in vivo
mutagenicity and clastogenicity studies, and no evidence indicated that sucralose have the
genotoxic potential to induce genetic effects [69].
In addition to sucralose, the DPD2544 strain was also inhibited by neotame, an artificial sweetener
with a very similar structure to aspartame but with higher sweetening power [
70
]. Toxicity of
neotame was previously reported, at doses higher than its admissible daily intake [
71
]. In this study,
the neotame concentrations tested were of lower concentrations but still induced TV1061 and inhibited
DPD2544 luminescences. The possible reason for these results is the capability of the bioreporter
bacteria to be affected by sub-active concentrations of the chemicals. It appears that some compounds
(e.g., antibiotics [
72
]), when used at sub-toxic concentrations, may activate or repress gene transcription,
which is distinct from their biological effects. Another possible reason is that the effective neotame
concentration in this study was still two-fold higher than in real food samples [
73
]. Other toxicity tests
did not evaluate the toxicity at such concentrations. The fact that neotame did not have any effect on
bacterial growth reinforced these findings.
Acesulfame K is one of the most used artificial sweeteners in the world and is “generally regarded
as safe” (GRAS) by the Food and Drug Administration of the United States of America [
71
]. However,
reports on the genotoxicity testing of ace-K are contradictory. It was found to be genotoxic and
clastogenic in mice [
74
]; non-mutagenic in a mammalian cell [
74
]; and non-cytotoxic and non-genotoxic
both in “
in vivo
” and “
in vitro
” experiments [
75
]. In this study, ace-K induced luminescence only in
genotoxic sensitive bacteria (DPD2794) indicating its possible genotoxicity.
As in the case of ace-K, aspartame also induced luminescence only with the genotoxicity sensitive
strain. Aspartame is a low-calorie sweetener used to sweeten a variety of low and reduced calorie
foods and beverages including low-calorie tabletop sweetener as well as in gums, breakfast cereals,
and other dry products [
76
]. Aspartame has been extensively evaluated for genotoxic effects in
microbial, cell culture and animal models [
77
,
78
]. These studies have shown evidence of induction of
chromosomal damage
in vitro
[
77
]. Indeed, in our case, DPD2794 strain, not only showed luminescence
induction effects but also showed dose-dependency (higher aspartame concentrations produced
Molecules 2018,23, 2454 10 of 20
stronger cells response) (Figure 1C). Thus, these results enforce the previous data on aspartame
genotoxicity to the E. coli strains.
In this study, amongst all of the tested artificial sweeteners, the strongest induction effect
was observed with saccharin (Table 1). Saccharin is the oldest chemical sugar substitute and the
best researched of all sweeteners [
79
], but it is still one of the most controversial food additives.
Many studies have shown that saccharin may act as a weak mutagen [
78
] or produce cytotoxic
effects [
80
]. Similar to these studies, our findings showed that saccharin induced the same luminescence
responses in both cytotoxic and genotoxic bacteria (Table 1). Nevertheless, TV1061 not only showed
much greater (three times more) responses than DPD2794, but also the growth rates of this strain was
inhibited (Figures 1and 2). The results suggest higher cytotoxic than genotoxic effects of saccharin
on the bacteria. In summary, two conclusions may be observed from these results. Firstly, different
artificial sweeteners exhibit different toxicity types of effects and create specific response patterns.
The second conclusion is that the bacterial responses were correlated to the results of previous toxicity
studies and bacteria may be used as a toxicity evaluation tool.
3.2. Sport Supplements Toxicity and Viability Effect
Nutrition has always been perceived as an integral component affecting physical performances in
sport competitions. The understanding of human metabolism and sport physiology shows a direct
correlation between the performance in sports and the manipulation in nutrient intake. Thus, during
the last decade, a large variety of sport supplements have been widespread and used routinely by
athletes. However, the side effects of these sport supplements have yet to be fully elucidated due
to the absence of compelling regulation and considerable variation in concentrations, terminology,
and combinations of these products. Nevertheless, a wide range of commercial sports supplements
are still available, with the majority of them containing artificial sweeteners. Numerous studies have
evaluated artificial sweeteners toxicity and their effect on the human health. However, in general,
they are concerned only with their addition into food products, with only a few examining their
effect on sports supplements. In this study, ten different commercially available sports supplements
were tested (Table 3). They were dissolved and exposed to the bioreporter bacterial panel, for toxicity
evaluation. Each sport supplement mixture contains a variety of different compounds, but all of
them include the addition of an artificial sweetener, either sucralose or/and ace-k, to sweeten the
supplement flavour.
Similar to the artificial sweeteners’ toxicity results, the bioreporter panel responded differently to
each tested sport supplement. Only when exposed to SS4, SS8, and SS10 mixtures did the bioreporter
panel show a similar response pattern (Table 2). Due to the complexity of the commercial mixture,
it is difficult to determine whether the responses were affected by the added artificial sweetener or
whether it is due to the presence of another component. However, it is still important to examine
their induction or inhibition effect on the bioreporter panel. For example, the bioluminescence of
DPD2544 strain was induced and inhibited by all supplements, while the inhibitory concentrations
were three-fold higher than the inducing concentrations. The DPD2544 strain exhibited a similar
inhibition effect when it was exposed to sucralose, the same artificial sweetener used in most of
the tested sport supplement mixtures (Table 1). However, the similarity in the response pattern to
the sport supplement SS6 (a mixture not containing sucralose), which has no visible effects on the
growth rates, indicates that the possible toxicity effect was produced by another component. Previously,
DPD2544 was used as a bioreporter for the determination of “general toxicity” of several environmental
contaminants, and its induction indicated interruptions in the fatty acid biosynthesis pathways [
53
].
The dose-dependent effect (induction at lower concentrations and inhibition at higher concentrations)
of the sport supplements on the DPD2544 strain (Figure 2) suggests the same cytotoxicity mechanisms.
Such effect was also observed with the TV1061 strain, a bacterium sensitive to general cytotoxic
damages. The sensitivity and reliability of this strain have been proven in many different applications
for air [
81
], soil [
82
] and water [
83
] toxicity monitoring. In this study, the induction effect of SS1, SS5,
Molecules 2018,23, 2454 11 of 20
SS7, and SS10 supplements on TV1061 indicates the activation of the cytotoxicity repair mechanisms in
the cells, and therefore provides data on their possible toxicity mechanisms. The greatest effect on the
bacterial panel was observed with the SS7 sport supplement mixture, where it not only induced and
inhibited light responses in all strains, but also decreased growth rates. Similar to previous results,
in all strains, the bioluminescence induction response pattern was observed at lower concentrations
than the inhibition response pattern. For example, the presence of SS7 induced bioluminescence at
three-fold or one-fold lower than the inhibitory concentrations in DPD2544 and TV1061, respectively
(Table 2). DPD2544 has shown similar response patterns for all the tested mixtures, even in cases where
no growth effects were observed (e.g., SS9). This strain is harboring the fusion lux genes with operon
fabA, a gene responsible for the formation of a double bond in fatty acids used in the membrane of
E. coli. Activation of this promoter is triggered by fatty acid starvation events caused by cell membrane
damages [
84
]. Thus, DPD2544 may be used as a tool for monitoring internal cellular mechanisms
that may be interrupted by consumption of sports supplements. The fact that the sport supplements
triggered bacterial responses without any effect on the cell growth rates also helps to determine their
toxicity grades (low in this case).
In contrast to DPD2544, cytotoxicity or genotoxicity effects (represented by growth and light
changes) were observed at much higher concentrations for all tested supplements in other bacteria
strains. For example, DPD2544 bacteria cells exposed to SS1, SS5, and SS8 were induced at nine-fold
lower concentrations than with that of the TV1061 strain. Interestingly, the same strains were induced
and inhibited simultaneously, while the growth rates of all cells were never increased or reduced in
the same sample. The kinetic responses of the DPD2794 and TV1061 were very similar, demonstrating
the same toxic pattern (Figure 2A,C). In both strains, the cells’ response patterns were observed only at
the highest tested concentration, while each sport supplement affected the bacterial response pattern
differently. For example, the luminescence signal in the SS5 and SS7 were inhibited and induced,
respectively. Such variations in the cell responses may be influenced by the differences in the sport
supplement composition, and indicates that both of these strains are sensitive to such changes. On the
other hand, DPD2544 have always demonstrated the same pattern in all of the tested sport supplements,
e.g., induction at the lower concentration and inhibition at the higher tested concentrations (Figure 2B).
The possible reasons for these uniform responses may be the presence of specific damaging agent/s in
all of the compositions that could have induced such an effect in this strain.
Table 3. Sport supplement profile.
Artificial
Sweeteners
Content
Recommended
Amount for
Consumption
(1 oz = 30 mL)
Ingredients
SS1 Sucralose
2 tablets (5 g),
recommended to
drink a lot of water
Creatine Hydrochloride, Cellulose, Dicalcium
phosphate, Enteric Coating (Cellulose, Sodium
Alginate, Medium Chain Triglycerides, Oleic
and Stearic Acid), Natural Mint Flavor,
Sucralose, Titanium Dioxide
SS2
Acesulfame
Potassium-K and
Sucralose
2 (7 g) to 8 (28 g)
scoops in 8–10 oz per
serving (2 scoops)
Black Tea Extract, Green Tea Extract, Green
Coffee Extract, Micronized Taurine, Micronized
L-Glutamine, Micronized L-Arginine,
Micronized L-Leucine, Beta-Alanine
(as CarnoSyn®), Micronized Citrulline,
Micronized L-Isoleucine, Micronized L-Valine,
Micronized L-Tyrosine, Micronized L-Histidine,
Micronized L-Lysine, Micronized
L-Phenylalanine, Micronized L-Threonine,
Micronized L-Methionine
Other Ingredients: Inulin, Acesulfame
Potassium, Citric Acid, FD&C Red #40, Malic
Acid, Natural and Artificial Flavors, Sucralose,
Silion Dioxide
Molecules 2018,23, 2454 12 of 20
Table 3. Cont.
Artificial
Sweeteners
Content
Recommended
Amount for
Consumption
(1 oz = 30 mL)
Ingredients
SS3
Acesulfame
Potassium-K and
Sucralose
1 (31 g) to 2 (62 g)
scoops in 6–8 oz
per scoop
Calcium, Cholesterol, Dietary Fibers,
Potassium, Protein, Saturated Fat, Sodium,
Sugars, Trans Fat
Other Ingredients: Acesulfame Potassium,
Cocoa (Processed with Alkali), Enzyme Blend
(Aminogen®, Lactase), Lecithin, Natural and
Artificial Flavors, Salt, Sucralose, Whey Protein
Blend (Whey Protein Isolate, Whey Protein
Concentrate, Whey Protein Hydrolysate),
Xanthan Gum
SS4 Sucralose
1 (31 g) to 2 (62 g)
scoops in 4–10 oz
per scoop
Calcium, Cholesterol, proteins, Sodium,
Saturated Fat, sugars, Trans Fat
Other Ingredients: Citric Acid, FD&C Red #40
Lake, Lactase, Sucralose, Natural and Artificial
Flavors, Soy Lecithin, Whey Protein Isolate,
Whey Protein Concentrate, Whey Peptides
SS5 Sucralose
2 (9 g) to 6 (27 g)
scoops in 10–12 oz
per serving
(2 scoops)
Caffeine, Green Tea Extract, Green Coffee
Extract, Micronized Taurine, Micronized
L-Glutamine, Micronized L-Arginine,
Micronized L-Leucine, Beta-Alanine
(as CarnoSyn®), Micronized Citrulline,
Micronized L-Isoleucine, Micronized L-Valine,
Micronized L-Tyrosine, Micronized L-Histidine,
Micronized L-Lysine HCI, Micronized
L-Phenylalanine, Micronized L-Threonine,
Micronized L-Methionine
Other Ingredients: Calcium Citrate, Calcium
Silicate, Citric Acid, Gum Blend (Cellulose
Gum, Xanthan Gum, Carrageenan), FD&C Blue
#2, FD&C Red #40, Inulin, Lecithin, Malic Acid,
Natural and Artificial Flavors, Silicon Dioxide,
Sucralose, Tartaric Acid
SS6 Acesulfame
Potassium-K
1 (29.4 g) rounded
scoop in 4–10 oz
Calcium, Protein, Saturated Fat, Sodium,
Sugars, Trans Fat
Other Ingredients: Acesulfame Potassium,
Aminogen®, Lactase, Lecithin, Natural and
Artificial Flavor, Whey Protein Isolate, Whey
Protein Concentrate, Whey Peptides
SS7
Acesulfame
Potassium-K and
Sucralose
1 (49 g) to 2 (98 g)
scoops in 6 oz
per scoop
Alpha lipoic acid, Calcium, Citric Acid,
Creatine Monohydrate, Creatine HCI,
Dicalcium Phosphate, Dextrose, L-alanine,
L-Isoleucine, L-Leucine, L-Valine, Magnesium
Oxide, Potassium, Sodium, Sugar, Taurine,
Vitamin B6, Vitamin C, Vitamin B12
Other Ingredients: Acesulfame-Potassium,
Dextrose, Ethyl-Cellulose, Glucose Polymers,
Modcarb™ [Oat Bran, Amaranth, Quinoa,
Buckwheat, Millet, Chia], Natural Flavors,
Calcium Silicate, Salt, Sucralose, FD&C Yellow
No. 6, Soy Lecithin, FD&C Yellow No. 5, Waxy
Maize (Corn Starch), (Cluster Dextrin)
Molecules 2018,23, 2454 13 of 20
Table 3. Cont.
Artificial
Sweeteners
Content
Recommended
Amount for
Consumption
(1 oz = 30 mL)
Ingredients
SS8
Acesulfame
Potassium-K and
Sucralose
1 (34 g) scoop in 6 oz
water or skim milk
Calcium, Cholesterol, Dietary Fiber, Iron,
Protein, Saturated Fat, Sodium, Sugar
Other Ingredients: Acesulfame-Potassium,
Alkalized Cocoa Powder, Calcium Carbonate,
Gum Blend (Cellulose Gum, Xanthan Gum,
Carrageenan), Natural and Artificial Flavors,
Salt, Soy Lecithin, Sucralose, Sunflower-based
Creamer (Sunflower oil, Corn syrup solids,
Sodium Caseinate, Mono-Diglycerides,
Dipotassium Phosphate, Tocopherols),
Tricalcium Phosphate, Whey Protein Isolate,
Whey Peptides, whey Protein Concentrate
SS9
Acesulfame
Potassium-K and
Sucralose
1 (32.4 g) to 2 (64.8 g)
scoops in 8–12 oz
Calcium, Cholesterol, Dietary Fiber, Iron,
Potassium, Protein, Saturated Fat, Sodium,
Sugar, Trans Fat, Vitamin A, Vitamin C
Other Ingredients: Acesulfame-Potassium,
Amino Matrix (L-Glycine, L-Taurine, BCAAs
(Leucine, Iso-Leucine, Valine), L-Glutamine),
Flax Seed Oil, Glucose Polymers, Lactase,
Natural and Artificial Flavors, Sucralose, Sea
Salt, Suspension Matrix (Xanthan Gum,
Cellulose Gum, Guar Gum), Whey Protein
Concentrate, Whey Protein Isolate, Whey
Protein Hydrolysate
SS10
Acesulfame
Potassium-K and
Sucralose
1 (34.9 g) to 2 (69.8 g)
scoops in 8–12 oz
Calcium, Cholesterol, Dietary Fiber, Iron,
Multi-level Amino Acid Growth Matrix,
Potassium, Protein, Saturated Fat, Sodium,
Trans Fat
Other Ingredients: Alanine, Arginine, Aspartic
Acid, BCAAs (L-Leucine, L-isoleucine,
L-Glutamine, L-valine), Cystine, Digestive
Enzyme Blend, Egg Albumen, Glycine,
Histidine, Lactase, Lysine, Methionine, Micellar
Casein, Partially-hydrolyzed Whey
Concentrate, Phenylalanine, Proline, Protease,
Serine, Tyrosine, Threonine, Tryptophan, Whey
Protein Isolate, Whey Protein Concentrate
4. Materials and Methods
4.1. Materials
LB Broth (L3022) Lennox L Broth (10 g/L Tryptone; 5 g/L Yeast Extract; 5 g/L NaCl); LB Broth
with agar (L2897) Lennox (Powder microbial growth medium); Kanamycin (K1876) disulfate salt
from Streptomyces kanamyceticus (amino-glycoside antibiotic), Sucralose
98% (HPLC) (69293),
Saccharin
99% (240931), Advantame (80054), Neotame (49777) and Acesulfame Potassium-K
(European Pharmacopoeia (EP) Reference Standard) (A0070000) were purchased from Sigma-Aldrich
(Sigma-Aldrich, St. Louis, MO, USA). Aspartame (47135) was purchased from SUPELCO (St. Louis,
MO, USA). Ethanol (absolute for analysis EMSURE
®
ACS, ISO, Reag, Ph Eur) was purchased from
Merck Millipore (Burlington, MA, USA) (1.00983.2500). A variety of ten sport supplements containing
artificial sweeteners were purchased from a local vendor. The concentrations range chosen for the pure
artificial sweeteners samples, was based on the FDA acceptable daily intake (ADI). ADI is calculated
as milligrams per kilogram body weight per day (mg/kg bw/d): ace-k (15), advantame (32.8),
aspartame (50), neotame (0.3), saccharin (15) and sucralose (5). In addition, for the sport supplements
samples, the recommended amount for consumption (as instructed by the company), is detailed in
Table 3, and was considered for the choice of the samples’ concentrations range.
Molecules 2018,23, 2454 14 of 20
4.2. Bioluminescent Bacteria from E. coli
The Escherichia coli strains used in this study, E. coli TV1061, DPD 2544 and DPD2794,
were obtained from S. Belkin (Hebrew University, Jerusalem, Israel) (see Table 4). The strains
harbor a plasmid-borne fusion of the different Promoters to a reporter gene [
85
]. The promoter
is chromosomally integrated to the reporter operon, which has five promotor-less structural genes
responsible for both the heterodimeric luciferase units (lux A and B) and the synthesis of the luciferase
substrate, tetradecanal, by an ATP-and NADPH-dependent multi-enzyme complex composed of fatty
acid reductase, transferase, and synthetase (lux C, D and E) [
53
]. The strain stocks were stored at
80
C with 20% (v/v) of glycerol, as a cell cryoprotectant additive. The bioreporter strains from
the stock solution were placed on LB-agar plates (10 g/L Tryptone; 5 g/L Yeast Extract; 5 g/L NaCl)
supplemented with 50
µ
g/mL kanamycin and, after incubation for two days at 37
C in an incubator
(Binder, Camarillo, CA, USA), they were stored at 4 C for future experiments.
Table 4. Bioluminescent bacterial strains.
Strain E. coli Host Strain Promoter Plasmid Stress Sensitivity Reference
TV1061 RFM443 grp E pGrpELux5 Heat Shock (Cytotoxic) [86]
DPD2544 W3110 fab A pFabALux6 Fatty Acid Availability (Cytotoxic) [53]
DPD2794 RFM443 rec A pRecALux3 SOS—DNA Damage (Genotoxicity) [87]
4.3. Growth Conditions
Bacterial cultivation prior to measurements was performed in 10 mL LB medium (10 g/L Tryptone;
5 g/L Yeast Extract; 5 g/L NaCl). Cells were grown overnight at 37
C in a shaking incubator (NB-205LF,
N-BIOTEK, SciMed (Asia) Pte Ltd., Singapore) at 120 rpm. Cultures were then diluted to approximately
10
7
cells/mL and re-grown in 10 mL LB at 30
C without shaking, until an early exponential phase
(Optical Density (O.D.) 600 nm of 0.2), as determined by a UVmini-1240, UV-VIS spectrophotometer
(Shimadzu, Singapore) (Figure 3).
4.4. Bioluminescence Assay
Bioluminescence activity was measured using a Luminoskan Ascent Luminometer (Thermo
Fisher Scientific, Waltham, MA, USA). Measurements took place in white 96-well microtiter plates
(NUNC) containing 90
µ
L of the bacterial culture at OD
600
= 0.2. Different concentrations of the tested
artificial sweeteners or sport supplements were added in volumes of 10
µ
L to each well (n= 3 for
each concentration). The negative control was obtained by adding 10
µ
L LB to the bacteria culture.
Moreover, positive control was obtained by the addition of: 2% (v/v) ethanol, 0.52 mM phenol and
800 ppb Mythomycin C as the known inducers for the following bacterial strains: TV1061, DPD2544
and DPD2794, respectively [
53
,
88
]. The artificial sweeteners or sport supplements concentrations
range used for the toxicity evaluation is dependent on the specific solubility properties of each tested
agent and is described in Table 1. During measurements (16 h), sample temperature was maintained at
26
C and the plates were continuously shaken. Luminescence values are presented in relative light
units (RLU) (Figure 3).
4.5. Growth Assay
The effect of the artificial sweeteners and sport supplements on the bacterial growth rates was
tested using the TECAN Infinite M200 PRO, City, Switzerland. Measurements took place in transparent
96-well microtiter plates (NUNC) containing 90
µ
L of the bacterial culture at OD
600
= 0.2. Different
concentrations of the tested artificial sweeteners or sport supplements were added in volumes of 10
µ
L
to each well (n= 3 for each concentration). The negative control was obtained by adding 10
µ
L LB to the
bacteria culture. Moreover, positive control was obtained by the addition of: 2% (v/v) ethanol, 0.52 mM
phenol and 800 ppb Mythomycin C as the known inducer for the following bacterial strains: TV1061,
Molecules 2018,23, 2454 15 of 20
DPD2544 and DPD2794, respectively [
53
,
88
]. During measurements (16 h), sample temperature was
maintained at 26
C and the plates were continuously shaken. Bacterial growth values are presented
in growth relative area AUC (under the curve) (GRA) (Figure 3).
Molecules 2018, 23, x FOR PEER REVIEW 15 of 20
volumes of 10 µL to each well (n = 3 for each concentration). The negative control was obtained by
adding 10 µL LB to the bacteria culture. Moreover, positive control was obtained by the addition of:
2% (v/v) ethanol, 0.52 mM phenol and 800 ppb Mythomycin C as the known inducer for the following
bacterial strains: TV1061, DPD2544 and DPD2794, respectively [53,88]. During measurements (16 h),
sample temperature was maintained at 26 °C and the plates were continuously shaken. Bacterial
growth values are presented in growth relative area AUC (under the curve) (GRA) (Figure 3).
Figure 3. Experimental process. (A) each bacteria strain tested was striked on an agar plate containing
Kanamycin, and incubated overnight at 37 °C ; (B) a starter was grown from a single colony from the
striked plate, and incubated overnight at 37 °C in a shaking incubator; (C) the starter was refreshed
by adding 200 μL of the overnight culture into 10 mL of fresh LB, and then grown for 34 h at 30 °C
in a non-shaking incubator; (D) the bacteria strains were then exposed to the different samples of
different concentrations in a high-throughput measurement using a 96-well plate; (E,F) the toxicity
(Relative Light Unit (RLU)) and growth (O.D. 600 nm) signals were measured continuously during
the 16 h incubation at 26 °C, in the Luminometer and TECAN reader, respectively.
4.6. Data Analysis
The bioluminescence signal relating the bacterial response to the different artificial sweeteners
and sport supplements was expressed as toxicity index (TI), which was calculated using the formula
TI = ((BS/BC)1), where BS is the average bioluminescent signal from the tested sample, either artificial
sweeteners or sport supplements, and BC is the average bioluminescent signal from the control. Based
on the results, a range of values was defined for better analysis of the toxicity effect: if TI ≥ 0.1, a toxic
inducing pattern is recognized, if TI ≤ −0.4, a toxic inhibiting pattern is recognized, and, if 0.4 < TI <
0.1, then no toxic effect is found. An additional two toxicity related parameters were determined as
follows: MLICMinimum Luminescent Inhibition Concentration; MLIndCMinimum
Luminescent Induction Concentration. Growth Relative AUC (area under the curve) (GRA) was
calculated using the following formula GRA = (GRAS/GRAC) × 100, where GRAS is the area under the
growth curve from the tested sample, either artificial sweetener or sport supplements and GRAC is
the area under the growth curve from the control. Based on the results, a range of values was defined
for better analysis of the growth effect: if GRA ≥ 120%, a growth inducing pattern is recognized, if
GRA ≤ 80%, a growth inhibiting pattern is recognized and, if 80% < GRA < 120%, then no growth
effect is found. An additional two growth related parameters were determined as follows: MGIC
Minimum Growth Inhibition Concentration; MGIndCMinimum Growth Induction Concentration.
5. Conclusions
Figure 3.
Experimental process. (
A
) each bacteria strain tested was striked on an agar plate containing
Kanamycin, and incubated overnight at 37
C; (
B
) a starter was grown from a single colony from the
striked plate, and incubated overnight at 37
C in a shaking incubator; (
C
) the starter was refreshed by
adding 200
µ
L of the overnight culture into 10 mL of fresh LB, and then grown for 3–4 h at 30
C in a
non-shaking incubator; (
D
) the bacteria strains were then exposed to the different samples of different
concentrations in a high-throughput measurement using a 96-well plate; (
E
,
F
) the toxicity (Relative
Light Unit (RLU)) and growth (O.D. 600 nm) signals were measured continuously during the 16 h
incubation at 26 C, in the Luminometer and TECAN reader, respectively.
4.6. Data Analysis
The bioluminescence signal relating the bacterial response to the different artificial sweeteners
and sport supplements was expressed as toxicity index (TI), which was calculated using the formula
TI = ((BS/BC)1)
, where B
S
is the average bioluminescent signal from the tested sample, either artificial
sweeteners or sport supplements, and B
C
is the average bioluminescent signal from the control. Based
on the results, a range of values was defined for better analysis of the toxicity effect: if TI
0.1,
a toxic inducing pattern is recognized, if TI
≤ −
0.4, a toxic inhibiting pattern is recognized, and,
if
0.4 < TI < 0.1
, then no toxic effect is found. An additional two toxicity related parameters were
determined as follows: MLIC—Minimum Luminescent Inhibition Concentration; MLIndC—Minimum
Luminescent Induction Concentration. Growth Relative AUC (area under the curve) (GRA) was
calculated using the following formula GRA = (GRA
S
/GRA
C
)
×
100, where GRA
S
is the area under
the growth curve from the tested sample, either artificial sweetener or sport supplements and GRA
C
is
the area under the growth curve from the control. Based on the results, a range of values was defined
for better analysis of the growth effect: if GRA
120%, a growth inducing pattern is recognized, if GRA
80%, a growth inhibiting pattern is recognized and, if 80% < GRA < 120%, then no growth effect is
found. An additional two growth related parameters were determined as follows: MGIC—Minimum
Growth Inhibition Concentration; MGIndC—Minimum Growth Induction Concentration.
Molecules 2018,23, 2454 16 of 20
5. Conclusions
The toxicity effect of six artificial sweeteners and ten sports supplements was evaluated by the
exposure to a bioreporter panel, which consists of three different bioluminescent bacterial strains
(E. coli), i.e., cytotoxic (TV1061), genotoxic (DP2794) and strain sensitive to membrane damage agents
(DPD2544). The differences in the cells’ response patterns did not only provide information about the
possible toxicity effect of these additives, but also allowed the creation of a specific response pattern
which may be used in future studies. Furthermore, the type of toxicity determined by the proposed
system was similar to the information found in literature, suggesting the efficiency of the proposed
system for fast and sensitive toxicity evaluation. Similarly, with the artificial sweeteners, the bioreporter
panel responded with different response patterns to the ten sports supplements tested in this study.
While some similarities were found in the cells’ responses to the artificial sweeteners, the complicated
sport supplements composition limit our understanding and information about the actual role of the
artificial sweetener addition. However, the triggered luminescent and affected growth rates indicate
that all tested sport supplements were toxic to the bacteria. The induction and inhibition effects on
the DPD2544 strain suggest that the primary mode of action of these mixtures was damaging the
cellular membrane. Moreover, E. coli is an indigenous gastro intestinal microorganism, and serves as
a model for the gut bacteria. The human colonic microbiome is a complex microbial community
that has a significant impact on individual’s health. This is a diverse community that reaches
high cell densities and includes dominant phyla including Bacteroidetes, Firmicutes, Actinobacteria
and Proteobacteria [
89
,
90
]. The indigenous gastrointestinal tract microflora has profound effects
on the anatomical, physiological and immunological development of the host [
91
]. In this study,
we demonstrated the toxicity effect on E. coli
in vitro
. With this consideration, we may speculate that
the response observed in our study may be relevant to gut microbiome and thus may influence human
health. Moreover, since artificial sweeteners are resistant to wastewater treatment processes [
33
],
they have been identified as emerging environmental pollutants [
31
,
32
]. Several environmental studies
have confirmed their distribution in the water cycle [
34
39
], with ace-k and sucralose concentrations
of up to the
µ
g L
1
range [
31
,
40
]. In this study, sucralose repressed luminescence in all the bioreporter
bacteria, the highest inhibition effect was observed with TV1061 strain (1 mg/mL), then with DPD2544
(50 mg/mL) and DPD2794 (100 mg/mL). In addition, ace-K induced luminescence only in genotoxic
sensitive bacteria (DPD2794) indicating its possible genotoxicity. The tested bioluminescent bacterial
panel can potentially be used for detecting artificial sweeteners in the environment.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions:
Conceptualization, A.K. and R.S.M.; Methodology, D.H. and E.E.; Validation, D.H. and
L.P.Y.; Formal Analysis, D.H., L.P.Y., A.K. and E.E.; Investigation, D.H., L.P.Y., F.C. and T.H.P.K.; Resources, A.K.,
A.I.Y.T. and R.S.M.; Data Curation, D.H. and L.P.Y.; Writing-Original Draft Preparation, D.H., L.P.Y. and E.E.;
Writing—Review and Editing, A.K., A.I.Y.T., R.S.M. and E.E.; Visualization, D.H., L.P.Y. and E.E.; Supervision,
A.K., A.I.Y.T., R.S.M. and E.E.; Project Administration, A.K., A.I.Y.T. and R.S.M.; Funding Acquisition, A.K., A.I.Y.T.
and R.S.M. All authors have read the final version of the manuscript.
Funding:
This publication is supported by the National Research Foundation (NRF) of Singapore under the
Campus for Research Excellence and Technological Enterprise (CREATE) and the Singapore-HUJ Alliance for
Research and Enterprise (SHARE), The Institute for Sport Research (ISR) and the Singapore International Graduate
Award (SINGA).
Acknowledgments: The authors thank Bharati Kadamb Patel for her work in establishing this study.
Conflicts of Interest:
The authors declared no potential conflicts of interest with respect to the research, authorship,
and/or publication of this article.
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Sample Availability: Samples of the compounds are not available from the authors.
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... In contrast to a growth-promoting effect, and in line with the observation that NAS are associated with a reduction in bacterial load in vivo (Figure 1) [38, 78,79,93,94], multiple in vitro studies demonstrate that NAS can directly inhibit bacterial growth (Figure 2) [79,88,[123][124][125][126][127][128][129]. Some of these effects may stem from NAS impacts on bacterial carbohydrate metabolism (Figure 2). ...
... Some of these effects may stem from NAS impacts on bacterial carbohydrate metabolism (Figure 2). Saccharin may interfere with microbial glucose transport, metabolism, and fermentation [126,[129][130][131] and was shown to modify expression of glucose transport and metabolism genes in ...
... Aspartame has also been shown to increase prophage induction in E. faecalis [136], while sucralose increases the mutation rate of E. coli in vitro [137]: both likely indicate activation of microbial stress response to NAS. Consistent with evidence for bacterial stress response, reactive oxygen species (ROS) and SOS-related stress genes are upregulated in response to AceK [134], and NAS-mediated induction of cellular stress was demonstrated in E. coli [129]. Thus, NAS appear to have several indirect effects on bacterial social behavior that may further alter gut microbiome dynamics through impacts on microbe-microbe interactions (Figure 2). ...
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Replacing sugar with non-nutritive artificial sweeteners (NAS) is a popular dietary choice for the prevention and management of metabolic syndrome and its comorbidities. However, evidence in human trials is conflicted regarding the efficacy of this strategy and whether NAS may counterintuitively promote, rather than prevent, metabolic derangements. The heterogeneity in outcomes may stem in part from microbiome variation between human participants and across research animal vivaria, leading to differential interactions of NAS with gut bacteria. An increasing body of evidence indicates that NAS can alter the mammalian gut microbiome composition, function, and metabolome, which can, in turn, influence host metabolic health. While there is evidence for microbiome-mediated metabolic shifts in response to NAS, the mechanisms by which NAS affect the gut microbiome, and how the microbiome subsequently affects host metabolic processes, remain unclear. In this viewpoint, we discuss data from human and animal trials and provide an overview of the current evidence for NAS-mediated microbial and metabolomic changes. We also review potential mechanisms through which NAS may influence the microbiome and delineate the next steps required to inform public health policies.
... Both the induced luminescent signals and bacterial growth were measured. The dose-dependent toxicity effect on E. coli in vitro was demonstrated [38]. In addition, Wang et al., (2018) evaluated the bacteriostatic effect of sucralose and saccharin on the growth of E. coli in liquid and solid media, finding that the ability to selectively inhibit the growth of enteric bacterial species may be due to inhibition of metabolic enzymes or alterations in nutrient transport [39,44,45]. ...
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Artificial sweeteners are additives widely used in our diet. Although there is no consensus, current evidence indicates that sucralose and saccharin could influence the gut microbiota. The aim of this study was to analyze the existing scientific evidence on the effects of saccharin and sucralose consumption on gut microbiota in humans. Different databases were used with the following search terms: sweeteners, non-caloric-sweeteners, sucralose, splenda, saccharin, sugartwin, sweet’n low, microbiota, gut microbiota, humans, animal model, mice, rats, and/or in vitro studies. In vitro and animal model studies indicate a dose-dependent relationship between the intake of both sweeteners and gut microbiota affecting both diversity and composition. In humans, long-term study suggests the existence of a positive correlation between sweetener consumption and some bacterial groups; however, most short-term interventions with saccharin and sucralose, in amounts below the ADI, found no significant effect on those groups, but there seems to be a different basal microbiota-dependent response of metabolic markers. Although studies in vitro and in animal models seem to relate saccharin and sucralose consumption to changes in the gut microbiota, more long-term studies are needed in humans considering the basal microbiota of participants and their dietary and lifestyle habits in all population groups. Toxicological and basal gut microbiota effects must be included as relevant factors to evaluate food safety and nutritional consequences of non-calorie sweeteners. In humans, doses, duration of interventions, and number of subjects included in the studies are key factors to interpret the results.
... Human studies failed to show a direct connection to cancer risk. However, other studies, , have shown association with kidney function decline and vascular risk factors (2) . ...
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Background: Non-nutritive sweeteners (NNSs) are becoming popular as sugar substitutes for diabetic patients or to decrease body weight. Aim of the work: This study was aimed to determine the effects of sucralose and sodium saccharin on some physiological parameters in male albino rats. Materials and methods: We used thirty male albino rats weighing from 100 to120 gm. The period of the experiment was 30 days. The animals were divided into three groups; Group 1: control, Group 2: rats received sucralose and group 3: rats received sodium saccharin. The following parameters were processed: serum glucose, ASAT, ALAT, serum creatinine, serum urea, protein and lipid profiles and hormonal levels (insulin, testosterone, serum T3 and T4). Results: There was an increase in ASAT and ALAT activities, serum creatinine and serum urea levels in group 2 and group 3, lipid profile in the group received sucralose (TC and HDL) and T3&T4 in the group received saccharin as compared to the control group. Meanwhile, a drop in serum glucose, insulin, total protein, albumin, albumin/globulin ratio and triglycerides in group 2 and group 3, lipid profile in the group received saccharin (TC and HDL) and T3&T4 in the group received sucralose was observed when compared to the control group. Conclusion: it could be concluded that sucralose and sodium saccharin must be carefully used because they have very dangerous effects-especially sodium saccharin-and we have to replace them with natural sugar.
... As concentrações de 1 e 100 mg/ml causaram citotoxicidade e genotoxicidade, respetivamente, em estirpes modificadas para detetar estes efeitos (25). Já em ratos (consumo ~3300 e ~1500 mg/kg pc/d nos grupos de dieta normal e HF, respetivamente, via água) observou-se que, não havendo diferenças no comportamento alimentar entre grupos, ...
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The use of sweeteners is a strategy for reducing sugar consumption. Sucralose is one of the most used, being described as safe by different reference entities, such as European Food Safety Authority and Food & Drug Administration. But since the publications by these entities are prior to 2017, it is important to update scientific knowledge. A search was made in the Pubmed database with the term “sucralose AND health”, for the last 5 years. The main topics addressed are routes associated with blood glucose management, weight gain, metabolic syndrome and intestinal microbiota, with results in general inconclusive. Studies are emerging that raise concerns about exposure in the uterus and possible insecurity of sucralose. Even so, based on current evidence, it´s not possible to assess that sucralose leads to negative health effects, when the ADI is respected. But its use/consumption should be moderate, since the long-term effects are not fully known.
... Vibrio fischeri (V. fischeri), representing the decomposer trophic level, is a standard acute toxicity model aquatic species and is widely used in the risk assessment of chemicals (Senko et al. 2019;Harpaz et al. 2018;Abbas et al. 2018; Min Li is willing to handle correspondence at all stages of refereeing and publication, also post-publication. Jurado et al. 2012;Ngwoke et al. 2021). ...
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Many quinoline (QL) derivatives are present in the environment and pose potential threats to human health and ecological safety. The acute toxicity of 30 haloquinolines (HQs) was examined using the photobacterium Vibrio fischeri. IC50 values (inhibitory concentration for 50% luminescence elimination) were in the range 5.52 to >200 mg·L−1. The derivative 5-BrQL exhibited the highest toxicity, with 3-ClQL, 3-BrQL, 4-BrQL, 5-BrQL, 6-BrQL, and 6-IQL all having IC50 values below 10 mg·L−1. Comparative molecular field analysis modeling based on the steric and electrostatic field properties of the HQs was used to quantify the impact of halogen substituents on their toxicity. QL derivative rings with larger substituents at the 2/8-positions and less negative charge at the 4/5/6/8-positions were positively correlated with acute toxicity towards V. fischeri.
... Its chemical composition is related to that of the carbohydrates. But, containing three chlorine atoms, it may present toxic effects like mutagenesis, carcinogenesis, provoke obesity and growth of glycosis levels [7][8][9][10][11][12]. Moreover, while stored in inappropriate way, it may form dioxines, even more toxic compounds [13][14]. ...
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A novel electroanalytical process for sucralose determination has been theoretically suggested. Sucralose is immobilized over an acridine derivative 9-9´-diacridyl, yielding a quaternary salt, which is thereby gradually electrochemically reduced to an N-N´-disubstituted acridone derivative. The correspondent mathematical model has been developed and analyzed by means of linear stability theory and bifurcation analysis, and this analysis has shown the high probability of the oscillatory and monotonic instabilities, due to the double electric layer structure changes during all of the stages of the process. Nevertheless, it also confirms the efficiency of 9-9´-diacridyl-modified electrode for sucralose determination in an electroanalytical system, which may be coupled with a fluorescent indicator.
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