Abstract derivatives are the true carcinogens in humans.


Polycyclic aromatic
hydrocarbons (PAHs) and their oxidized derivatives are widespread in the atmospheric particulate matter, water, and soil
in urban areas. Therefore, inhaled or dietary intake of PAHs and PAHs
derivatives are the main exposure pathway for humans, especially for the
children living in these areas since they have more ingestion and dermal
pathway risks than adults do. Although parent PAHs have been extensively
studied, limited research has been done regarding the biological effects of
PAHs derivatives which have been shown more likely to be dissolved and more
mobile in the environment. Moreover, oxidized PAHs derivatives are the true
carcinogens in humans. Nevertheless, by applying metabolomics analysis approaches
as well as various mass spectrometry and big data analysis methods, we attaining
a better understanding of biological effects of PAHs and their metabolites in humans. In this review, the biological effects determinations
of PAHs and oxidized PAHs derivatives
by applying metabolomics analysis approach are briefly

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Among persistent organic
pollutants (POPs), PAHs constitute a large and diverse chemical category,
produced by natural and anthropogenic sources. PAHs molecules are composed of
two or more fused benzene rings and may include alkyl, nitrogen or oxygen
substituents in derivatives. In recent years, due to human activities, such as
the burning of fossil fuels and car exhaust, environmental concentrations of
PAHs in many industrialized and developing countries are rapidly increasing
(Shen et al., 2013). Increases in the PAHs emissions could be hazardous to
human health, especially for young children and the developing fetus (Perera et al., 2012). In addition, inhalation
exposure to these compounds may induce cardiac dysfunction, mortality in the
uterus, growth retardation and lower intelligence (Elie et al., 2015).

Most PAHs in the
environment result from incomplete combustion and pyrolysis processes of
organic carbon, including biomass, petroleum, and coals. Based on their
origins, PAHs can come from natural and anthropogenic sources Natural sources
include oil seeps from crude oil deposits, forest fires, volcanoes and erosion
of ancient sediment. For example, some PAHs such as perylene are produced
naturally from the biochemical transformation of organic carbon. Anthropogenic
PAHs are formed either by thermal alteration of organic carbon or its
incomplete combustion (Gan et al., 2009). Today, the major sources of PAHs in
the environment are from the human utilization of petroleum products and
incomplete combustion of fossil fuels, biofuels or other forms of organic
carbon, far exceeding natural sources. Based on their formation process, PAHs
from both natural and anthropogenic sources can be classified into three
groups: pyrogenic, petrogenic, and biogenic. Pyrogenic PAHs result from
incomplete combustion of fossil fuels and biomass under high temperatures. They
are released in the form of exhaust and solid residues, thereby ubiquitous in
soils and sediments. Petrogenic PAHs originate from petroleum products such as
crude oil, coal, and gasoline and are formed under relatively low temperatures
during fossil fuel formation processes. Direct spillage from petroleum is of
course also a common petrogenic PAH source. In most cases, pyrogenic PAHs
dominate over petrogenic PAHs due to human impact. Petrogenic PAHs are
introduced into the environment through accidental oil spills, release from tanker
operations, and municipal runoff. Biogenic PAHs are produced during degradation
of vegetative organic substances by plants, algae, and microorganisms. In
addition, they are produced during the slow transformation of organic carbon by
plants and microorganisms (Abdel-Shafy et al.,

A large number of PAHs
environmental toxicity studies have focused on parent PAHs because of their
potential mutagenic and carcinogenic properties. However, other studies have
shown that oxygenated PAHs
derivatives (oxy-PAHs) have negative effects on human health (Lundstedt et al,
2007). These oxy-PAHs together with their parent compounds are produced during
the incomplete combustion of organic compounds. After that, they are released
into the atmosphere (Ringuet et al., 2012). Unlike PAHs, these derivatives are
not monitored by any government agencies or international organizations.
However, their physical and toxic properties are significant for further
research. Oxy-PAHs in the environment are
more mobile than their parent PAHs because they have higher water solubility.
In addition, the particulate PAHs or their oxygen derivatives found in diesel
exhaust are the latest suspected of the main driving factors of cardiovascular,
neural degeneration and lung diseases (Elie et al., 2015). Since limited
research has been done on the toxicity pathways regarding parent PAHs and their
derivatives, more research in terms of fully understanding the toxic effects of
PAHs and oxy-PAHs and their mechanism are
urgently needed.

metabolomics are relatively new techniques to evaluate the biological consequences
of the exposures of chemical molecules (Lankadurai et al., 2013). Metabolite models can be used to
characterize the alteration from chemicals such as PAHs or oxy-PAHs to toxic reactions
endpoints that are caused by PAHs. Metabolomics analysis can be targeted, where
known metabolites are quantified, or untargeted, where a comprehensive analysis
is performed of all known and unknown metabolites. The untargeted method allows
for any significant differences in the pattern of graphical depiction and it usually provides
information about toxicity mechanism, pathways, and possible exposure
biomarkers (Dumas et al., 2014). Among the most commonly used instruments in
the study of metabolomics analysis, liquid chromatography-mass spectrometry
(LC-MS) provides a strong platform for the metabolites of biology and
environment disturbance identification since it has various advantages such as
fast analysis, quantitative, and multiplex (Chen and Kim, 2013). In addition,
when combined with genomics approaches, its function can be amplified, as a
method to connect genetic variants and phenotypic traits (Adamski and Suhre,


PAHs aquatic
metabolomics study on zebrafish

The main sources of PAHs in water bodies are atmospheric
particulate matter deposition, polluted groundwater runoff, industrial
wastewater, urban wastewater discharges and oil spills on rivers and lakes.
Since PAHs have low solubility and tend to adsorb particulate matter, they are
usually found in water at low concentrations. Some of the PAH concentrations
measured in aquatic systems include rivers, pounds, seawater, wastewater and
urban runoff (Latimer and Zheng, 2003). PAHs tend to accumulate in sediments
rather than water (Juhasz and Naidu, 2000). The concentrations of PAHs in
specific sediments can range from ppm to ppb, depending on how close the area
is to PAHs sources such as industry, cities and water streams. In North
America, the total PAH concentration in marine sediments is usually in the
range of 2.17-170,000 ppb. Sediment core studies have shown an increase in PAHs
over the past 100-150 years (Latimer and Zheng, 2003).

Although a large number of organisms have been used in
metabolomics studies, the zebrafish model has been applied insufficiently. As a
developmental vertebrate model, zebrafish metabolic analysis has a unique
advantage as an in vivo animal model.
A lot of fish anatomy and physiology are highly homologous to the those of mammals.
Moreover, there is a considerable amount of genetic identity with humans in
zebrafish, with about 87% similarity. In addition, zebrafish embryos developed
rapidly and remained transparent in many organogenesis,
which makes the researchers able to
perform large-scale and high-throughput screening at a lower cost (Lieschke and
Currie, 2007). Recent research shows zebrafish may be an ideal reference model
system for performance metabolomics-related studies. In further, metabolic
changes in zebrafish are conserved in human samples (Santoro, 2014).

Some embryos zebrafish
transcription and heredity research has been conducted to assess developmental
toxicity PAHs and oxy-PAHs (Goodale et al., 2013; Jayasundara et al., 2014).
However, the current research is lack of metabolic information. Compare
metabolic disturbances with gene transcription and gene alterations protein
expression, produced by PAHs and oxy-PAHs exposures, it will be a significant
step towards clarifying the mechanism toxicity. Therefore, more study is needed
to define the effects of PAHs and oxy-PAHs on culture in zebrafish by applying
non-targeted metabolomics methods. By combining in vivo metabolic profiles with multivariate variables pattern
recognition and pathway analysis, metabolomics data found that PAHs and
oxy-PAHs exposures to be strongly associated with changes that are known to
affect protein biosynthesis, mitochondrial dysfunction (oxidative stress),
neurodevelopment, interference Vascular development and cardiac development
(heart toxicity). Based on the pathway database and previous pathway research
on the zebrafish metabolome, it was discovered that PAHs exposures are
responsible for the effects of
glutathione metabolism; glycine, serine
and threonine metabolism; cysteine and
methionine metabolism; purne metabolism;
phenylalanine metabolism; phenylalanine,
tyrosine and tryptophan metabolism; aminoacyl-tRNA biosynthesis (Elie et al., 2015).


PAHs metabolism and human
health effects

Some active metabolites
of PAHs such as epoxide and dihydrogen diol have posed one of the major health
problems. They can combine with cellular proteins and have the potential of
producing toxic effects on DNA even if the parent PAHs are not detected in the
analysis (Armstrong et al., 2004). The effects of the destruction of biochemical targets and cell damage can
result in mutations, developmental abnormalities, tumor and cancer (Bach et al,
2003). A mixture of PAHs has more harmful effects than individual PAHs to
humans in terms of cancer. According to the U.S. Environmental Protection
Agency (USEPA, 2008), seven PAHs compounds have been classified as probable
human carcinogens: benz(a)anthracene,
benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, chrysene, dibenz(ah)anthracene, and indeno(1,2,3-cd)pyrene.
Figure 1 simply shows the flow chart connecting both short-term and long-term PAHs
exposure and health effects (Kim et al., 2013).

Figure 1. Short and long term health effects of PAHs exposure (Kim et al., 2013).

PAHs are hydrophobic
compounds that can be easily transported across the cell membrane by passive
diffusion. After diffusing into the lung cells, the parent PAHs molecules are
thought to be carcinogens because they do not directly induce DNA damage
(Alexandrov et al., 2010). In fact, the conversion of a single polycyclic
aromatic to its oncogenic metabolite causes the cause of cancer. The
transformation of these compounds involves a variety of metabolic enzymes in
three known major pathways: CYP1A1 / 1B1 and epoxide hydrolase (CYP / EH
pathway), CYP peroxidase pathway and aldo-keto
reductase pathway (AKR). Often, PAHs are involved in CYP enzyme metabolism and some other
metabolic conversions to phenol, catechol,
and quinone, and form oxides, free-radical cations or reactive quinones. They
all react with DNA to produce DNA adducts. For example, quinones can react with
guanine N-3 and guanine N-7 in DNA (Liu, 2002). DNA adducts can lead to the
formation of DNA replication mismatches, changes in methylated promoters (Yang
et al., 2012), leading to mutations or aberrant gene expression that eventually
lead to tumorigenesis.

Benzo(a)pyrene (BaP) is
one of the most carcinogenic PAHs. Figure 2 shows the conversion from BaP to
(BPDE), the final carcinogen to be DNA adducted (Moorthy et al., 2015).

Figure 2. Major pathways
of metabolic activation of BaP to DNA-binding metabolites (Moorthy et al., 2015).

Biomarkers of PAHs metabolomics

The objective of our
research is to understand the relationship between PAHs and humans in the
environment, to assess the environmental impact of PAHs on human activities and
to assess the impact of various aspects of PAHs on human health. A major
challenge in studying the impact of environmental PAH exposure on human health
is to determine the causal relationship between the extent of exposure to PAHs
and the prevalence of various biological endpoints of adverse events such as cancer
and irritation. This causal relationship can only be established if every
element on the source-contact-dose continuum is connected. For adverse health
consequences of exposure to PAHs, chemicals must be released from the source,
transported through the environmental media, reached the body’s receptors, into
the body, and accumulated to a sufficient degree within the target tissue in an
organism. Eventually, the
adaptation mechanism is down, resulting in changes in adverse health outcomes.

Researchers have
developed a variety of methods to assess PAHs exposures to the environment and workplace
to internal levels of PAHs. In many studies, pyrene
metabolites, such as 1-hydroxypyrene,
have been widely used as urinary biomarkers for PAH exposure (Sobus et al.,
2009). Most importantly, pyrene is present in relatively high concentrations
(2-10%) of PAHs in all mixtures. In some environments, pyrene concentrations in
total PAHs are generally constant (McClean et al., 2012). However, 1-hydroxypyrene cannot
always be used to predict exposure to BaP or other carcinogenic PAHs because
the relative concentrations of pyrene and BaP may vary widely (Srogi, 2007).

It should be noted that
the concentration or excretion of parent PAHs compounds or metabolites in body
fluids or urine is not only dependent on external exposure but also on the
absorption, biotransformation, and excretion, which can be significantly
different among individuals. BaP-DNA adduct in peripheral lymphocytes and with
proteins such as albumin have also been used as indicators of reactive
metabolites. As binding of electrophilic PAHs metabolites to DNA is thought to
be a key step in the initiation of cancer, measurement of DNA adducts could be
an indicator of PAHs exposure and also of the dose of the ultimate reactive
metabolite (Perera et al., 2011).


of PAHs metabolomics study

spectrometry is widely used as a metabolomics analysis platform because it
provides high sensitivity, reproducibility, and versatility. It measures
molecules and the mass of fragments to confirm their identity. This information
is gained by measuring the mass?to?charge ratio (m/z) of ions that are formed
by inducing the loss or gain of a charge from a neutral species. A complex
mixture of a sample containing metabolites can be directly measured using a
separation method such as liquid chromatography and gas chromatography and then
analyzed by mass spectrometry.
Direct injection has been successfully used in high flux metabolomics. However,
due to the hundreds of thousands of ions that can exist in metabolomics
experiments, before entering the mass spectrometer,
it is recommended to use
chromatographic separation to minimize signal suppression and allow
for greater sensitivity. In addition, use of the retention time can further
help metabolite identification. In addition to the m/z and the retention time
information, ion recognition is promoted by the fracture pattern, which can be
obtained through tandem mass spectrometry (Johnson et al., 2016).

The study by Wang et al.,
(2015) used an LC-MS metabolomics method
combined with multivariate statistical data analysis to investigate human body
metabolic disturbances after PAHs exposure. This was achieved by analyzing the
urine samples of a large population of children and elderly people living in an
area polluted by the coking industry and
a non-polluted control area. Metabolic alterations in response to PAHs exposure
were evaluated to discover potential metabolic biomarkers. In addition, a sensitive
liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for
measuring nine urine metabolites of PAHs to assess the level of exposure to the
same group of PAHs. Finally, the individual PAHs exposure and its metabolic
consequences were assessed by correlation analysis to determine dose-effect
relationships. The whole research strategy is shown in Figure 4 (Wang et al.,
2015). This can be considered as a typical procedure for metabolomics study of PAHs

Figure 4. Long-term PAHs
exposure environment of general population research by metabolomics method
based on LC-MS (Wang et al., 2015).


analysis of PAHs metabolomics study

Because metabolomics generate large data sets, computational tools
for processing and interpretation are very important. Since big data processing,
statistical analysis, metabolite identification and biological explanation
related problem are not trivial, there are now tools (e.g., automation) that
accelerate computational workflows and provides a user-friendly tool for both
beginners and professional scientists. The development of chemical information
tools, which are used for the calculation of metabolomics results can
effectively support the experimental data upload, processing, statistical
analysis and identification of metabolites, and when combined with
bioinformatics tools, can put metabolites in a biological environment.
Metabolomics analysis, especially not-targeted metabolomics, can lead to very
complicated data sets. They contain
information on thousands of ions that are generated in the mass spectrometer
from each sample, in which the ions represent the precursor intact metabolite
or its fragments, adducts or isotopes. Therefore, applying
computing tools to reduce the redundancy of these complex datasets and to identify the most relevant
metabolites is very important (Wolf et al., 2010; Johnson et al., 2015, 2016).

Multivariate techniques
are most widely used in big data analyses of PAHs metabolomics studies such as principal
components analysis (PCA), partial least squares-discriminant analysis
(PLS-DA), and orthogonal PLS-DA (OPLS-DA). In addition, volcano plots can be
useful to show fold change versus significance as p-value, and heat maps can
show the alteration of various metabolomes among the different groups (Elie et al., 2015; Wang et al., 2015).

Elie et al., (2015)
established a non-targeted metabolomics method to determine the effects of 4 ?M
of benzaanthracene (BAA) and benzaanthracene-7,12-dione (BAQ) on zebrafish
metabolic function. Through the integration of multivariate, single variable
and pathway analysis, a total of 63 metabolites were significantly changed
after 5 days of exposure. Obvious disturbance shows that BAA and BAQ affect
protein biosynthesis, mitochondrial function, neural development, vascular
development, and cardiac function. As shown in Figure 5, PCA-DA and PLS-DA
models of two-dimensional chart showed that BAA and BAQ groups and the control
group differ in PC1 from each other.
In addition, as shown in Figure 6, the change of comparison between the control
group and group BAQ, metabolites increased slightly.

Figure 5. PLS-DA score plot (A) and PCA-DA score plot (B) in the
positive ion mode based on the normalized data (Elie et al., 2015).

Figure 6. Heat map produced by hierarchical clustering of the most
significantly different metabolites obtained from the positive ion mode. The
log 2 fold change in metabolite levels is color-coded: red pixels denote up-regulation;
blue pixels denote down-regulation. Fold changes were based on peak intensities
and relative to a pooled average sample from the control group (Elie
et al., 2015).

Wang et al., (2015) used
the metabolomics method based on LC-MS in order to study various levels of PAHs
exposure in terms of human urinary metabolic alteration. As a result, compared
with individuals between the exposure group and the control group, 18
metabolites related to amino acids, purine and lipid metabolism significantly
changed. These findings suggested that chronic environmental exposure to low
levels of PAHs in the human body would cause oxidative stress effects. In
addition, 1-hydroxyphenanthrene and dodecadienylcarnitine are potentially sensitive
and reliable biomarkers for PAHs exposure in the general population. The study
demonstrates that a metabolomic approach is a useful tool to identify the
various metabolic changes of environmental PAHs exposure in the general population
and provides new insight into the mechanisms underlying PAHs-induced toxic
effects. The OPLS-DA scatter plot in Figure 7 showed that the exposed group
could be clearly separated from the control group based on the 1400 peaks
detected by LC-MS.

Figure 7. OPLS-DA score
plots based on the (A) UHPLC?(+)ESI?MS and (B) UHPLC?(?)ESI?MS data from
elderly non-smokers. Blue dots, elderly
nonsmokers in the control group (n = 96); red dots, elderly nonsmokers in the
exposed group (n = 142) (Wang et al., 2015).


We are living in an
environment in which PAHs and their derivatives are ubiquitous. Due to their
toxic, carcinogenic, and mutagenic nature, it is significant to study their
biological effects and corresponding mechanisms in humans. As discussed in this
review, metabolomics analysis approach is a powerful tool for achieving these
goals, especially by utilizing zebrafish model in the aquatic system. The research
results which presented in this review demonstrate the utility of LC-MS based metabolomics in combination with the
developmental zebrafish model to provide deeper mechanistic insights into the
link between chemical exposure and the profound impact of organisms. Applying
this approach to more PAHs and diverse PAHs can greatly extend the metabolomics
used to predict the structure-activity relationships and potential hazards of
various and ubiquitous contaminants. Moreover, by applying various big data
analysis methods, people can gain a better understanding of how PAHs and their
derivatives alter biochemical pathways to cause adverse health effects. These all
will provide significant information for toxicology and molecular biology


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