Natalija Atanasova-Pancevska1* and Aleksandra Markovska2
1Department of Microbiology and Microbial Biotechnology, North Macedonia
2Quality Consulting Macedonia, North Macedonia
*Corresponding author: Natalija Atanasova Pancevska, Department of Microbiology and Microbial Biotechnology, North Macedonia
Submission: September 20, 2021;Published: November 29, 2021
Volume9 Issue5November, 2021
Additives are a group of organic and inorganic compounds that are not raw materials and are used in the production of food in order for the products to be of better quality or longer lasting, to protect the taste, or to improve the taste or appearance. Some of the additives have been used for centuries, for example by salting meat or using CO2 in wine. The main groups of food additives are antioxidants, colors, flavor enhancers, sweeteners, emulsifiers and stabilizers and preservatives. The FAO has also given a definition of additives, according to which additives are substances that are intentionally added to products, usually in small quantities, have no nutritional value, and the purpose of their addition is to improve the appearance, smell, taste, consistency or durability of the product. Some of the additives that are not approved by the European Commission are approved and used in Australia and New Zealand. Given all of the above, it is easy to conclude that aspects of food safety will be the number one topic in this century. The practical outcome of this review is presented as a set of recommendations for future research in this area. The use of the data in this review is proposed as a training set to develop the framework into a diagnostic tool.
Keywords: Additives; Interaction; Food safety; Combination
The use of chemical additives in food is a problem that has been actively considered
for a long time. The modern world has established mechanisms for approving additives,
but there are still additives for which different parts of the world have different views. The
importance of interactions of food additives with other components of food (i.e., nutrients
and non-nutrients) has been assessed and certain aspects of toxicology included. With the
latest example, the fatal death of a child, from a combination of additives, a new topic is slowly
emerging, a new field of work, determining combinations of foods that are beneficial to human
health and combinations of foods that are not beneficial to consumer health. A topic that will
require an opinion, generally accepted throughout the world.
The Regulation provides for : a) Community 1 lists of approved food additives which are
set out in Annex II and III of the Regulation; b) Conditions of use for food additives used in
foods, including in food additives, food enzymes as covered by Regulation (EC) No. 1332/2008,
food flavorings as covered by Regulation (EC) No.1334/2008 and nutrients; c) rules on the
labelling on food additives sold as such; d) specific rules on the “carry-over” principle; e) rules
on the la-belling of the so called “Southampton colors”. f) specifications (purity criteria) to be
established for permitted food additives.
How are they controlled?
All additives are thoroughly assessed for safety before they are permitted for use, and they
are only then permitted to be used in a limited range of products and in certain amounts. These amounts are based on an Acceptable Daily Intake (ADI) calculated
by the European Food Safety Authority (EFSA) from the results of
safety tests. The ADI represents an amount that can be ingested daily
over a lifetime without appreciable health risk. Approved additives
are given a number, and some are also awarded an ‘E’. An E shows
the additive has been accepted as safe for use within the European
Union. Even when an additive has been approved, regular repeat
testing is required to maintain its status as ‘approved’. Food labels
give information about most additives present in the ingredients
list, so that consumers can make informed choices.
Some parents report that artificial colors and preservatives
trigger hyperactivity in their children, although randomized
controlled trials have generally failed to demonstrate a link.
However, a study published in 2007 [1] suggested that mixes of
certain artificial colors used in foods and drinks together with the
preservative sodium benzoate, are associated with hyperactivity
in some children, although it is not yet clear whether this is the
cause of the hyperactivity. These artificial colors are Sunset yellow
(E110), Tartrazine (E102), Carmoisine (E122) Ponceau 4R (E124),
Quinoline yellow (E104) and Allura red (E129). Food and drink
containing any of these six colors must carry a warning on the
packaging. This will say ‘May have an adverse effect on activity and
attention in children’. The FSA encourage manufacturers to work
towards finding alternatives to these colors. Some manufacturers
and retailers have already taken action to remove them.
The FAO has also given a definition of additives, according to which additives are substances that are intentionally added to products, usually in small quantities, have no nutritional value, and the purpose of their addition is to improve the appearance, smell, taste, consistency or durability of the product. Some of the additives that are not approved by the European Commission are approved and used in Australia and New Zealand. Over the last 30 years in main focus of food safety science is food additive-additive chemical interactions with appropriate relevant information on food additive-food component interactions.
Interactions
One of the major concerns about the safety of dietary supplement ingredients is that interactions between a supplement and other ingested substances (e.g., drugs, other dietary supplements,1 conventional foods) will result in adverse clinical outcomes due to an increase or decrease in the level of the dietary supplement in the organism, an increase or decrease in the level of other xe-nobiotics,2 or combined toxicities. Potential adverse clinical outcomes may result if a dietary supplement lowers a drug’s effective concentration. Such a drop in active drug concentration can have serious consequences, especially for persons whose health depends on the therapeutic effects of a drug. Many dietary supplement products are mixtures of two or more substances, some of unknown structure, making an evaluation of interactions more complex, but also more likely to be of clinical concern as they are consumed simultaneously. Interactions can be detected with human, animal, or in vitro studies or predicted on the basis of how related substances behave.
Types of interactions
There are numerous mechanisms for interactions among xenobiotics, but most can be categorized as direct chemicalchemical, pharmacodynamic, or pharmacokinetic interactions.
Direct chemical-chemical interactions
The formation of chemical-chemical complexes can modify the action of one or both chemicals. In general, these types of interactions require ingestion of both chemicals within a relatively short time of each other. An example of a direct chemical-chemical interaction occurs in the small intestine, where calcium carbonate taken as a supplement may bind to an acid substance, such as the antibiotic tetracycline, to form an insoluble product. In this case, since the acid was a drug, the action of the drug would be reduced or lost. Other examples include cholestyramine, which adsorbs other drugs, thereby decreasing their availability for absorption, and antacids, which can block iron or zinc uptake. In addition to forming complexes, antacids may significantly change the rate of absorption of other chemicals by altering gastric pH or gastric emptying time, depending on the extent to which pH affects the amount of chemical in the un-ionized state [2].
Interactions with dietary supplements
There are examples of pharmacodynamic interactions that have been noted with dietary supplement ingredients. The antihypertensive effect of guanabenz acetate (a drug used for hypertension) is due to its central agonistic α-2-adrenoceptor activity [3]. Thus, concomitant consumption of yohimbine bark, which contains an α-2-adrenoreceptor antagonist, may diminish the antihypertensive activity of guanabenz through its opposing pharmacodynamic effect. Another example is between the inotropic drug digitalis [4] and Hawthorne leaf or flower; data suggest that both the Hawthorne leaf and the flower may also have a positive inotropic and electrophysiological effect on the heart Schwinger et al. [5]. If digitalis and the hawthorne leaf or flower are taken together, the additive response may be excessive and lead to a serious adverse event [5]. Another additive effect would be exhibited by the ginkgo leaf if its purported antagonism of plateletactivating factor occurred; if ingested with a cyclooxygenase inhibitor, such as aspirin, an in-creased propensity for bleeding would occur [6].
Pharmacokinetic interactions
Pharmacokinetic interactions are interactions that occur when one substance affects the absorption, distribution, metabolism, or excretion of another substance, resulting in altered levels of one of the substances or its metabolites. These interactions include effects caused by the chemicals on xenobiotic metabolizing enzymes and transporters that affect the time course of the concentration of one or both of the chemicals in the body. These interactions commonly take place in the intestines, liver, or kidney and are further categorized based on their site of action.
Altered metabolism
Interactions that alter metabolism warrant attention. Xenobiotics often undergo extensive metabolic alteration by enzymes, resulting in the formation of structurally modified derivatives (metabolites) that may possess different pharmacologic activities (either greater or less) when com-pared with that of the consumed parent compound. There are more than 30 families of xenobiotic metabolizing enzymes in humans, many of which may be limiting for biotransformation of the consumed xenobiotic. If an ingested xenobiotic increases or decreases the amount or activity of a given enzyme, its own rate of metabolism may be altered, as well as that of other consumed compounds. The clinical effect of changes in enzyme metabolism rates will depend on the xenobiotic(s) involved and their metabolites and potencies.
An important group of xenobiotic metabolizing enzymes are the cytochrome P450 (CYP) enzymes, a superfamily of hemoproteins that mediate the biotransformation of 7endogenous and exogenous compounds in the liver, as well as in the intestine and elsewhere. Some CYP isozymes found to be involved with significant pharmacokinetic reactions in humans are CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4.In addition, CYP2A6 and CYP2B6 are involved in metabolizing certain xenobiotics (Health Canada, 2000). Since many chemicals are substrates for the same CYP isozymes, one compound may inhibit the activity of the enzyme metabolizing another compound that is ingested concomitantly. In addition, ingestion of a chemical hours before another chemical may induce the production of more enzyme or inhibit normal enzyme synthesis, thus affecting the rate of metabolism of a second chemical metabolized by that same enzyme. While not with-out controversy, grapefruit juice provides one example of an interaction associated with CYP enzymes; it is reported to suppress CYP3A4 and change the concentration of drugs metabolized by the enzyme. When considering dietary supplement ingredient safety, assays for xenobiotic alterations of enzyme metabolism may generate important signals of possible concern, as discussed below.Altered absorption, distribution, and excretion
Until recently, pharmacokinetic interactions were considered as primarily attributable to the effects on xenobiotic metabolizing enzymes. However, an increasing number of transporters that affect chemical absorption, distribution, and excretion now seem to also play a significant role in pharmacokinetic interactions [7]. Transporters regulate the flux of substances into and out of cells or perform a variety of transmembrane transport functions. Depending on their location and activity, they may have a significant effect on the concentration of a chemical at its site of action. Interactions between chemicals resulting from competition at transporters are not uncommon. Thus in vitro methods to evaluate the effect of chemicals on particular transporters have been developed. Due to differences in human and animal transporters, the methods often employ hu-man transporter proteins expressed in artificial in vitro systems, enabling the detailed study of human transporter protein functions with regard to drugs and other xenobiotic substances, including dietary supplement ingredients.
Effects on Excretion
Renal or biliary excretion of xenobiotics, and thus the steadystate plasma concentration of xenobiotics, may also be affected by other xenobiotics. Changes in renal clearance of one xenobiotic can occur through effects of another substance on the urinary pH. Another mechanism for inter-action is the effect of one substance on the active secretion of another substance into the renal tubule. Methods to evaluate the effects of a xenobiotic on excretion are available; they include measurement of tubular uptake, such as perfused kidney assays, or assays at the cellular level. Dietary supplement ingredients that inhibit tubular uptake or in any other way disrupt molecular mechanisms important to excretion of other xenobiotics should be considered of potential concern.
Predicting the potential of ingredients to cause pharmacokinetic interactions
Techniques currently available allow the determination of the extent to which one substance may impact the concentration of other concomitantly ingested substances. There are numerous wellaccepted in vitro assays designed specifically to determine if a drug may interact with other sub-stances. There are also approaches for describing structures of chemicals likely to cause interactions. These in vitro studies and other approaches have focused on determining which drugs affect metabolizing enzymes and transporters and could similarly be used to determine which dietary supplements may lead to interactions. Whether an interaction predicted on the basis of in vitro studies actually occurs clinically will depend on whether the dietary supplement compound attains a concentration in vivo adequate to reproduce the effect observed in vitro, as discussed in more detail below.
Invitro prediction of pharmacokinetic effects
In vitro studies for determining which xenobiotics affect
transporters and metabolic enzymes ideally employ human
transporter proteins or human metabolic enzymes. For example,
subcellular fractions of human liver tissue are commonly used, as
are whole-cell models such as isolated hu-man hepatocytes, liver
slices, and cell lines derived from human cancer cells. Human
transporters and enzymes can also effectively be studied by
expressing them in other cell types. Changes in either the activity or
amount of enzyme or transporter are detected with activity assays,
pharmacological assays, and immunochemical or mRNA assays that
detect changes in protein or transcription.
In vitro assays for predicting possible interactions are a wellaccepted
staple of the drug development process. The limitation to
using these assays to predict clinical interactions lies, like most in
vitro assays, in relating the dose at which enzyme or transporter
effects are observed with the amount of unbound xenobiotic present
at the active site in vivo. If information about the concentration of
xenobiotic reached in vivo is available, a comparison of a dietary
supplement ingredient’s inhibitory binding constants (Ki) for the CYP enzymes and the in vivo concentration (Cmax) may place the in
vitro information in the appropriate perspective.
Animal and human in vivo data in predicting pharmacokinetic effects
Given the inter- and intraspecies differences in xenobiotic
metabolizing enzymes, it is ideal to study xenobiotic metabolism
using human cells, subcellular fractions of human tissue, or
heterologous expressed human proteins, although information
about effects on animal proteins may serve as a preliminary
indicator of concern. The study of human proteins in transgenic
animals may im-prove ability to relate effects observed in animals
or animal cells to humans.
Humans themselves may also be studied to determine if a given
xenobiotic may cause an observable interaction. Such tests are
usually designed to compare the levels of a test substrate with and
without the xenobiotic in question. For example, a study of St. John’s
wort in humans demonstrated that it increased the metabolism of
CYP3A4 substrates. Even if specific interaction assays are not done,
information about the in vivo concentrations achieved in humans is
useful in placing in vitro information in perspective.
Databases for Predicting Interactions
Databases helpful for identifying substances likely to interact with other substances have been organized. For example, the database produced by the University of Washington is useful for locating information about potential interactions of particular dietary supplements with other sub-stances. The database also organizes information, such as drug effects on CYP enzymes, that may be useful for identifying potential interactions between particular drugs and supplements. A publicly available website at the Indiana University School of Medicine provides information about drugs metabolized by specific P450 isoforms.
Vulnerable subpopulations
Some individuals are particularly sensitive to adverse effects
from xenobiotic interactions be-cause of polymorphic differences
that affect the metabolism of some xenobiotics. There are
recognized genetic polymorphisms that account for diminished or
absent expression of one or more forms of xenobiotic-metabolizing
enzymes. There are documented adverse effects directly resulting
from the altered metabolism of certain drugs metabolized by these
enzymes. A well-known example is people who exhibit little or no
CYP2D6 activity in the liver because of inherited genes defective
in expression of this form of CYP-a condition that affects 7 to 10
percent of Caucasians, by one estimate. As a result, such individuals
are found to experience toxic effects from ordinary doses of the
antihypertensive agent debrisoquine, as well as many other drugs
for which metabolic elimination is primarily catalyzed by CYP2D6.
It would be reasonable to expect that any dietary supplement
ingredient dependent on CYP2D6 for metabolic conversion could
potentially produce toxic effects in such persons. Numerous
other polymorphisms in xenobiotic metabolism have been or are
being identified. Such data can serve to identify people who may
be particularly sensitive to dietary supplements cleared by these
polymorphic xenobiotic metabolizing systems. Published work on
sulfur dioxide and ascorbic and nitrous acid reactions with other
food additives to form stable compounds. In some cases, such as
between nitrite and sorbic acid, the compounds formed have a
potentially higher toxicity than the original additives. No adverse
effects have been demonstrated in real foods, however, probably
due to the adoption of substantial safe-ty margins between noeffect
levels in animals and the maximum levels of additives to
which humans could be exposed.
The reactions discussed in this review are those most likely to
occur in current additive usage. However, due to the large numbers
of permitted food additives, many more interactions occur in foods
that could lead to chemical reactions under favorable conditions.
Food additives are widely used for technological purposes and
their presence is often substantial daily diet. They have also been
accused of various toxic reactions in humans. The toxicity of the
food color tartrazine, the preservatives sodium nitrate and sodium
benzoate, and the antioxidant BHT, was studied using the protozoan
Tetrahymena pyriformis as a toxicological model. The 4 food
additives were added to Tetrahymena cultures and DNA content of
the protozoan nuclei measured by an image analysis system. These
food additives caused a statistically significant in-crease in DNA
content suggesting stimulation of the mitotic process. This system
may contribute to the investigation of the cellular action of food
additives, since mitogenic stimuli substantially alter susceptibility
to chemical carcinogenesis.
Breast Cancer Resistance Protein (BCRP), multidrug resistance
associated protein 2 (MRP2) and P-Glycoprotein (P-GP) are ABC
transporters that are expressed in the intestine, where they are
involved in the efflux of many drugs from enterocytes back into
the intestinal lumen. The inhibition of BCRP, MRP2, and P-GP can
result in enhanced absorption and exposure of substrate drugs.
Food additives are widely used by the food industry to improve
the stability, flavor, and consistency of food products. Although
they are considered safe for consumption, their interactions with
intestinal transporters are poorly characterized. Therefore, in this
study, selected food additives, including preservatives, colorants,
and sweeteners, were studied in vitro for their inhibitory effects on
intestinal ABC transporters. Among the studied compounds, several
colorants were able to inhibit BCRP and MRP2, whereas P-GP was
fairly insensitive to inhibition. Additionally, one sweetener was
identified as a potent inhibitor of BCRP. Dose–response studies
revealed that the IC50 values of the inhibitors were lower than
the estimated intestinal concentrations after the consumption of
beverages containing food colorants. This suggests that there is
potential for previously unrecognized transporter-mediated food
additive-drug interactions.
Different foods possess different bioactive compounds with
varied antioxidant capacities. When foods are consumed together,
the total antioxidant capacity of food mixtures may be modified
via synergistic, additive, or antagonistic interactions among these
components, which may in turn alter their physiological impacts. Eleven foods from three categories, including fruits (raspberry,
blackberry, and apple), vegetables (broccoli, tomato, mushroom,
and purple cauliflower), and legumes (soybean, adzuki bean,
red kidney bean, and black bean) were combined in pairs. Four
assays (total phenolic content, ferric reducing antioxidant power,
2,2-diphenyl-1-picrylhydrazyl, radical scavenging capacity, and
oxygen radical absorbance capacity) were used to evaluate the
antioxidant capacities of individual foods and their combinations.
The results indicated that within the same food category, 13, 68,
and 21% of the combinations produced synergistic, additive, and
antagonistic interactions, respectively, while the combinations
produced 21, 54, and 25% synergistic, additive, and antagonistic
effects, respectively, across food categories. Combining specific
foods across categories (e.g., fruit and legume) was more likely
to result in synergistic antioxidant capacity than combinations
within a food group. Combining raspberry and adzuki bean extracts
demonstrated synergistic interactions in all four chemical-based
assays. Compositional changes did not seem to have occurred in the
mixture. Results in this study suggest the importance of strategically
selecting foods or diets to maximum synergisms as well as to minimum
antagonisms in antioxidant activity.
Exposure to non-nutritional food additives during the critical
development window has been implicated in the induction
and severity of behavioral disorders such as Attention Deficit
Hyperactivity Disorder (ADHD). Although the use of single food
additives at their regulated concentrations is believed to be
relatively safe in terms of neuronal development, their combined
effects remain unclear. We therefore examined the neurotoxic effects
of four common food additives in combinations of two (Brilliant
Blue and L-glutamic acid, Quinoline Yellow and aspartame) to
assess potential interactions. Mouse NB2a neuroblastoma cells
were induced to differentiate and grow neurites in the presence
of additives. After 24 h, cells were fixed and stained, and neurite
length measured by light microscopy with computerized image
analysis. Neurotoxicity was measured as an inhibition of neurite
outgrowth. Two independent models were used to analyze
combination effects: effect additivity and dose additivity. Significant
synergy was observed be-tween combinations of Brilliant Blue
with L-glutamic acid, and Quinoline Yellow with aspartame, in both
models. Involvement of N-methyl-D-aspartate (NMDA) receptors
in food additive-induced neurite inhibition was assessed with a
NMDA antagonist, CNS-1102. L-glutamic acid- and aspartameinduced
neurotoxicity was reduced in the presence of CNS-1102;
however, the antagonist did not prevent food color-induced
neurotoxicity. Theoretical exposure to additives was calculated
based on analysis of content in foodstuff, and estimated percentage
absorption from the gut. Inhibition of neurite outgrowth was found
at concentrations of additives theoretically achievable in plasma
by ingestion of a typical snack and drink. In addition, Trypan Blue
dye exclusion was used to evaluate the cellular toxicity of food
additives on cell viability of NB2a cells; both combinations had a
straightforward additive effect on cytotoxicity. These data have
implications for the cellular effects of common chemical entities
ingested individually and in combination. The association of
food additives with hyperactivity is a popularly accepted notion.
Feingold hypothesized that food dyes are pharmacologically active
substances that induce or aggravate symptoms of hyperactivity in
children. Subsequent studies have confirmed that food colors can
induce clinical symptoms of hyperactivity and can also alter brain
electrical activity in a subgroup of children with ADHD.
Yet there is still no conclusive scientific evidence to indicate
that any of the currently available food additives have any adverse
effect on human development. The present study investigated the
developmental neurotoxic effects of four common food additives.
Two independent models were used to assess interactions in
this study: “effect additivity” and “dose additivity.” Combinations
acted synergistically in reducing the length of neurite outgrowth
from differentiating mouse NB2a neuroblastoma cells. Quinoline
Yellow and aspartame showed greater synergy than Brilliant
Blue and L-glutamic acid; however, the results indicate that both
combinations are potentially more toxic than might be predicted
from the sum of their individual compounds.
The colors examined in this study are synthetic dyes that are
certified as safe and are permitted for use as food additives in
the U.K. Brilliant Blue (E133) is banned in the majority of the EU
countries and causes mitochondrial toxicity in vitro. The use of
Quinoline Yellow (E104) in foods is banned in Australia, Norway,
and the U.S., and genotoxic effects have been reported [8]. However,
very little information about the neurotoxicity of food colors is
available, and the mechanism by which they exert their toxic effect
on nerve cells is not clear.
In contrast, the Excitatory Amino Acids (EAA), L-glutamic
acid, and aspartic acid are well established neurotoxins. Over
three decades ago, it was discovered that L-glutamic acid destroys
dendrites and cell bodies of neurons in the developing brain, thus
causing brain lesions. Oral and subcutaneous administration of
L-glutamic acid to infant animals (rodents and primates) induces
acute neuronal necrosis in several regions of the developing brain
including the hypothalamus and the hippocampus. As adults,
treated animals show stunted skeletal development, obesity, and
female sterility. Retinal neuronal changes also occur in rats after
prolonged administration of high L-glutamic acid diets, whilst in
adult humans, it elicits headache in susceptible individuals and is
believed to be responsible for the “Chinese Restaurant Syndrome”
symptoms of which include chest pain, numbness, burning and
facial pressure.
Similar hypothalamic lesions can be induced by aspartic
acid, one of two of the constituent amino acids in the dipeptide
sweetener aspartame. Following ingestion, aspartame is rapidly
hydrolyzed to release three biologically active chemicals: aspartic
acid, phenylalanine, and methanol, which are absorbed into the
portal blood. It has been commonly used in diet drinks and sugarfree
foods throughout the world for over 20 years, despite reports
of panic attacks, seizures, and headaches with its use. Recently,
chronic exposure of aspartame was found to affect memory in rats
[9].
Excitotoxins destroy central neurons by excessive stimulation
of postsynaptic excitatory mem-brane receptors whereas the
under-stimulation of such receptors during the developmental
period triggers apoptosis. Thus, excitotoxic and apoptotic
neurodegeneration are two distinct cell death processes that are
readily distinguishable ultra-structurally. It is well established that
an excitotoxic mechanism plays a role in many neurologic disorders,
from acute insults such as stroke and head trauma to chronic
neurodegenerative states such as Huntington’s disease and the
acquired immunodeficiency syndrome (AIDS) dementia complex.
The over-stimulation of such receptors leads to the opening of
voltage-dependent calcium channels, initiating a cascade of events
in-volving the activation of protein kinases, phospholipases,
proteases, Nitric Oxide Synthase (NOS), generation of free radicals
and mitochondrial damage. The NMDA receptor plays a prominent
role because of its high permeability to Ca2+; however other EAA
receptor subtypes also contribute to these processes. Selective
non-competitive NMDA antagonists such as MK-801 markedly
protect CNS neurons against direct excitotoxic effects; this has
been demonstrated in primary cultures of hippocampal neurons
following L-glutamic acid exposure. Our data are consistent with
a role for excitotoxicity in the mechanism of injury caused by some
flavor-enhancing food additives. CNS-1102 (a NMDA receptor
antagonist) protected against both L-glutamic acid and aspartameinduced
neurite inhibition, whilst the results demonstrated that
food color-induced neurotoxicity was not mediated by NMDA
receptor activation. When assessing cell death mechanisms of food
additive combinations, we found that both combinations studied
had a straightforward additive effect on cell viability, as measured
by Trypan Blue dye exclusion. The mechanisms of synergistic
neurotoxicity are therefore unrelated to effects on viability.
The list of non-nutritional additives in foods is extensive, and
it is virtually impossible to hold a single chemical responsible for
a particular dysfunction. For many of the commercial products
analyzed, more than one additive was detected. Children’s sweets
were found to contain both Brilliant Blue and Quinoline Yellow,
whilst corn snacks were found to contain both aspartame and
L-glutamic acid. Humans are not only exposed to such simple
mixtures, but also to complex mixtures of chemicals rather than
to individual chemicals, yet they continue to be tested for toxicity
in isolation from each other. Also present in the environment are
numerous potentially neurotoxic compounds such as pesticides
that get into foods somewhere along the chain from farm to plate.
It has been estimated that we have in our bodies between 300
and 500 chemicals that did not exist 50 years ago. Thus, mixture
studies are important to elucidate whether these interactions or
chronic exposure to such mixtures would cause deleterious effects
to a developing child. Very few long-term experiments have been
attempted, and cumulative toxic effects have hardly been explored
at all.
Despite being a major factor relevant to clinical settings,
combination pharmacology is a topic that has not received much
attention. It is essential that such investigations are carried out
by reliable experimental procedures and appropriate statistical
methods; however, there is widespread disagreement over
terminology, definitions, and models for the analysis of interactions.
Several methods for calculating the expected combination effect
of two or more compounds are currently in use, the majority
of which can be associated with two popular basic concepts
known as effect additivity and dose additivity. Effect additivity
focuses on measuring the effects of mixtures at only one specified
concentration for each compound, thus lacking the information
on concentration response relationships. Dose additivity is an
equally valid procedure for analyzing interactions between agents
irrespective of their mechanisms of action and aims to establish
the re-quired concentrations of individual compounds within a
combination that produces a specified level of effect. However, this
method requires tedious testing with a variety of concentrations
for the determination of each data point on the is bologram, where
a vast amount of information is eventually lost. Furthermore, is
bolographic analysis requiring independent statistical analysis,
which can be extremely complicated. There is no generally accepted
agreement as to which of the two concepts is more appropriate;
therefore, we have attempted to carry out this study using both
models to confirm our findings. Similar conclusions could be drawn
from both methods.
During the developmental period of synaptogenesis (brain
growth spurt period), neurons are very sensitive to specific
disturbances in their synaptic environment [10]. In humans, this
period ex-tends from the sixth month of gestation to several years
after birth, thus children are considerably more vulnerable to harm
from toxic chemicals than adults. Since they are at a crucial stage of
development, exposure to toxic chemicals may directly or indirectly
attack their undeveloped nervous, immune, and endocrine systems.
Dysfunction in any of these systems may lead to deleterious
health effects. Cell proliferation, migration, differentiation, and
synapse formation progress in a tightly programmed and orderly
fashion. Interference with any stage of this cascade of events may
alter normal progression of subsequent stages and short-term
disruptions may have long-term effects later in life. Neurotoxicants
may interfere with brain development and subsequent function at
exposure levels that have minimal or no effect on the adult brain.
The in vitro cell line may of course be more susceptible to toxicity
than an in vivo model. Specifically, the in vitro neurotoxicity assay
has no representation of the blood-brain barrier (BBB); however,
this is not complete in the developing human brain until around
six months after birth. Furthermore, some regions of the brain are
not protected by a BBB at any time in life [10]; thus they remain
in contact with any potentially neurotoxic substances circulating in
the blood. Such regions are known as the Circumventricular Organs
(CVOs), which include the portal system of the hypothalamus. The
CVOs make up a minor proportion of the brain but are functionally
very important regions.
For the measurement of potential body concentrations
following ingestion, a number of assumptions have been made in
our calculations. Absorption and distribution of additives need to be taken into account when relating in vitro data to in vivo effects,
however there is little information about the absorption from the
gut in infants or their distribution in the brain. In all cases, the
potential whole body volume exposure to individually assessed
additives lies within the range that we found to reduce neurite
outgrowth by approximately 10-20% for Quinoline Yellow, 40-50%
for Brilliant Blue and aspartame, and 50-60% for L-glutamic acid.
Furthermore, neurite outgrowth would be reduced significantly
more if the compounds were assessed in combination.
In conclusion, we present evidence that specific combinations
of common food additives show synergistic effects to inhibit
neuronal cell differentiation in vitro, using both the effect additivity
and dose additivity models of assessing interactions. The immature
nervous system may be vulnerable to such toxic insults since this
marker of neurotoxicity was found at concentrations of additives
theoretically achievable in plasma by ingestion of a snack and/or
drink typically consumed by children. Mechanisms of synergistic
toxicity have yet to be determined, and the implications of these
data on developmental disorders remain to be investigated.
Dietary supplements have a potential to adversely affect public health by interacting with other substances. Whether this concern is addressed by labeling precautions, withdrawal of such dietary supplements from the market or requiring warning labels related to usage with other xenobiotics is a regulatory decision. Pharmacists and physicians are made aware of drugs and foods that can potentially interact with other drugs, and drug labeling warns about potential problems. There is no analogous prescribed mechanism to prevent dietary supplement-mediated interactions. A number of pieces of information can suggest a possible interaction between a dietary supplement ingredient and other substances. The potential seriousness of these interactions varies and is placed in perspective by considering if a particular interaction leads to serious adverse events and the likelihood that the interaction will occur.
© 2021 Natalija Atanasova-Pancevska. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and build upon your work non-commercially.