Abstract derivatives are the true carcinogens in humans.

AbstractPolycyclic aromatichydrocarbons (PAHs) and their oxidized derivatives are widespread in the atmospheric particulate matter, water, and soilin urban areas. Therefore, inhaled or dietary intake of PAHs and PAHsderivatives are the main exposure pathway for humans, especially for thechildren living in these areas since they have more ingestion and dermalpathway risks than adults do. Although parent PAHs have been extensivelystudied, limited research has been done regarding the biological effects ofPAHs derivatives which have been shown more likely to be dissolved and moremobile in the environment. Moreover, oxidized PAHs derivatives are the truecarcinogens in humans. Nevertheless, by applying metabolomics analysis approachesas well as various mass spectrometry and big data analysis methods, we attaininga better understanding of biological effects of PAHs and their metabolites in humans.

In this review, the biological effects determinationsof PAHs and oxidized PAHs derivativesby applying metabolomics analysis approach are brieflydiscussed. IntroductionAmong persistent organicpollutants (POPs), PAHs constitute a large and diverse chemical category,produced by natural and anthropogenic sources. PAHs molecules are composed oftwo or more fused benzene rings and may include alkyl, nitrogen or oxygensubstituents in derivatives. In recent years, due to human activities, such asthe burning of fossil fuels and car exhaust, environmental concentrations ofPAHs in many industrialized and developing countries are rapidly increasing(Shen et al., 2013). Increases in the PAHs emissions could be hazardous tohuman health, especially for young children and the developing fetus (Perera et al.

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, 2012). In addition, inhalationexposure to these compounds may induce cardiac dysfunction, mortality in theuterus, growth retardation and lower intelligence (Elie et al., 2015). Most PAHs in theenvironment result from incomplete combustion and pyrolysis processes oforganic carbon, including biomass, petroleum, and coals.

Based on theirorigins, PAHs can come from natural and anthropogenic sources Natural sourcesinclude oil seeps from crude oil deposits, forest fires, volcanoes and erosionof ancient sediment. For example, some PAHs such as perylene are producednaturally from the biochemical transformation of organic carbon. AnthropogenicPAHs are formed either by thermal alteration of organic carbon or itsincomplete combustion (Gan et al., 2009). Today, the major sources of PAHs inthe environment are from the human utilization of petroleum products andincomplete combustion of fossil fuels, biofuels or other forms of organiccarbon, far exceeding natural sources. Based on their formation process, PAHsfrom both natural and anthropogenic sources can be classified into threegroups: pyrogenic, petrogenic, and biogenic. Pyrogenic PAHs result fromincomplete combustion of fossil fuels and biomass under high temperatures.

Theyare released in the form of exhaust and solid residues, thereby ubiquitous insoils and sediments. Petrogenic PAHs originate from petroleum products such ascrude oil, coal, and gasoline and are formed under relatively low temperaturesduring fossil fuel formation processes. Direct spillage from petroleum is ofcourse also a common petrogenic PAH source. In most cases, pyrogenic PAHsdominate over petrogenic PAHs due to human impact.

Petrogenic PAHs areintroduced into the environment through accidental oil spills, release from tankeroperations, and municipal runoff. Biogenic PAHs are produced during degradationof vegetative organic substances by plants, algae, and microorganisms. Inaddition, they are produced during the slow transformation of organic carbon byplants and microorganisms (Abdel-Shafy et al.,2015).

A large number of PAHsenvironmental toxicity studies have focused on parent PAHs because of theirpotential mutagenic and carcinogenic properties. However, other studies haveshown that oxygenated PAHsderivatives (oxy-PAHs) have negative effects on human health (Lundstedt et al,2007). These oxy-PAHs together with their parent compounds are produced duringthe incomplete combustion of organic compounds. After that, they are releasedinto the atmosphere (Ringuet et al., 2012). Unlike PAHs, these derivatives arenot monitored by any government agencies or international organizations.However, their physical and toxic properties are significant for furtherresearch.

Oxy-PAHs in the environment aremore mobile than their parent PAHs because they have higher water solubility.In addition, the particulate PAHs or their oxygen derivatives found in dieselexhaust are the latest suspected of the main driving factors of cardiovascular,neural degeneration and lung diseases (Elie et al., 2015). Since limitedresearch has been done on the toxicity pathways regarding parent PAHs and theirderivatives, more research in terms of fully understanding the toxic effects ofPAHs and oxy-PAHs and their mechanism areurgently needed.Environmentalmetabolomics are relatively new techniques to evaluate the biological consequencesof the exposures of chemical molecules (Lankadurai et al., 2013).

Metabolite models can be used tocharacterize the alteration from chemicals such as PAHs or oxy-PAHs to toxic reactionsendpoints that are caused by PAHs. Metabolomics analysis can be targeted, whereknown metabolites are quantified, or untargeted, where a comprehensive analysisis performed of all known and unknown metabolites. The untargeted method allowsfor any significant differences in the pattern of graphical depiction and it usually providesinformation about toxicity mechanism, pathways, and possible exposurebiomarkers (Dumas et al., 2014). Among the most commonly used instruments inthe study of metabolomics analysis, liquid chromatography-mass spectrometry(LC-MS) provides a strong platform for the metabolites of biology andenvironment disturbance identification since it has various advantages such asfast analysis, quantitative, and multiplex (Chen and Kim, 2013).

In addition,when combined with genomics approaches, its function can be amplified, as amethod to connect genetic variants and phenotypic traits (Adamski and Suhre,2013).  PAHs aquaticmetabolomics study on zebrafishThe main sources of PAHs in water bodies are atmosphericparticulate matter deposition, polluted groundwater runoff, industrialwastewater, urban wastewater discharges and oil spills on rivers and lakes.Since PAHs have low solubility and tend to adsorb particulate matter, they areusually found in water at low concentrations. Some of the PAH concentrationsmeasured in aquatic systems include rivers, pounds, seawater, wastewater andurban runoff (Latimer and Zheng, 2003). PAHs tend to accumulate in sedimentsrather than water (Juhasz and Naidu, 2000).

The concentrations of PAHs inspecific sediments can range from ppm to ppb, depending on how close the areais to PAHs sources such as industry, cities and water streams. In NorthAmerica, the total PAH concentration in marine sediments is usually in therange of 2.17-170,000 ppb. Sediment core studies have shown an increase in PAHsover the past 100-150 years (Latimer and Zheng, 2003).Although a large number of organisms have been used inmetabolomics studies, the zebrafish model has been applied insufficiently. As adevelopmental vertebrate model, zebrafish metabolic analysis has a uniqueadvantage 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 inzebrafish, with about 87% similarity. In addition, zebrafish embryos developedrapidly and remained transparent in many organogenesis,which makes the researchers able toperform large-scale and high-throughput screening at a lower cost (Lieschke andCurrie, 2007). Recent research shows zebrafish may be an ideal reference modelsystem for performance metabolomics-related studies.

In further, metabolicchanges in zebrafish are conserved in human samples (Santoro, 2014).Some embryos zebrafishtranscription and heredity research has been conducted to assess developmentaltoxicity PAHs and oxy-PAHs (Goodale et al., 2013; Jayasundara et al., 2014).However, the current research is lack of metabolic information. Comparemetabolic disturbances with gene transcription and gene alterations proteinexpression, produced by PAHs and oxy-PAHs exposures, it will be a significantstep towards clarifying the mechanism toxicity. Therefore, more study is neededto define the effects of PAHs and oxy-PAHs on culture in zebrafish by applyingnon-targeted metabolomics methods. By combining in vivo metabolic profiles with multivariate variables patternrecognition and pathway analysis, metabolomics data found that PAHs andoxy-PAHs exposures to be strongly associated with changes that are known toaffect protein biosynthesis, mitochondrial dysfunction (oxidative stress),neurodevelopment, interference Vascular development and cardiac development(heart toxicity).

Based on the pathway database and previous pathway researchon the zebrafish metabolome, it was discovered that PAHs exposures areresponsible for the effects ofglutathione metabolism; glycine, serineand threonine metabolism; cysteine andmethionine metabolism; purne metabolism;phenylalanine metabolism; phenylalanine,tyrosine and tryptophan metabolism; aminoacyl-tRNA biosynthesis (Elie et al., 2015). PAHs metabolism and humanhealth effectsSome active metabolitesof PAHs such as epoxide and dihydrogen diol have posed one of the major healthproblems. They can combine with cellular proteins and have the potential ofproducing toxic effects on DNA even if the parent PAHs are not detected in theanalysis (Armstrong et al., 2004). The effects of the destruction of biochemical targets and cell damage canresult in mutations, developmental abnormalities, tumor and cancer (Bach et al,2003).

A mixture of PAHs has more harmful effects than individual PAHs tohumans in terms of cancer. According to the U.S. Environmental ProtectionAgency (USEPA, 2008), seven PAHs compounds have been classified as probablehuman 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 PAHsexposure and health effects (Kim et al., 2013). Figure 1. Short and long term health effects of PAHs exposure (Kim et al.

, 2013).PAHs are hydrophobiccompounds that can be easily transported across the cell membrane by passivediffusion. After diffusing into the lung cells, the parent PAHs molecules arethought to be carcinogens because they do not directly induce DNA damage(Alexandrov et al., 2010). In fact, the conversion of a single polycyclicaromatic to its oncogenic metabolite causes the cause of cancer. Thetransformation of these compounds involves a variety of metabolic enzymes inthree known major pathways: CYP1A1 / 1B1 and epoxide hydrolase (CYP / EHpathway), CYP peroxidase pathway and aldo-ketoreductase pathway (AKR). Often, PAHs are involved in CYP enzyme metabolism and some othermetabolic conversions to phenol, catechol,and quinone, and form oxides, free-radical cations or reactive quinones. Theyall react with DNA to produce DNA adducts.

For example, quinones can react withguanine N-3 and guanine N-7 in DNA (Liu, 2002). DNA adducts can lead to theformation of DNA replication mismatches, changes in methylated promoters (Yanget al., 2012), leading to mutations or aberrant gene expression that eventuallylead to tumorigenesis.Benzo(a)pyrene (BaP) isone of the most carcinogenic PAHs. Figure 2 shows the conversion from BaP toBP-7,8-dihydrodiol-9,10-epoxide(BPDE), the final carcinogen to be DNA adducted (Moorthy et al., 2015). Figure 2. Major pathwaysof metabolic activation of BaP to DNA-binding metabolites (Moorthy et al.

, 2015).Biomarkers of PAHs metabolomicsstudyThe objective of ourresearch is to understand the relationship between PAHs and humans in theenvironment, to assess the environmental impact of PAHs on human activities andto assess the impact of various aspects of PAHs on human health. A majorchallenge in studying the impact of environmental PAH exposure on human healthis to determine the causal relationship between the extent of exposure to PAHsand the prevalence of various biological endpoints of adverse events such as cancerand irritation. This causal relationship can only be established if everyelement on the source-contact-dose continuum is connected.

For adverse healthconsequences of exposure to PAHs, chemicals must be released from the source,transported through the environmental media, reached the body’s receptors, intothe body, and accumulated to a sufficient degree within the target tissue in anorganism. Eventually, theadaptation mechanism is down, resulting in changes in adverse health outcomes.Researchers havedeveloped a variety of methods to assess PAHs exposures to the environment and workplaceto internal levels of PAHs. In many studies, pyrenemetabolites, 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 intotal PAHs are generally constant (McClean et al., 2012). However, 1-hydroxypyrene cannotalways be used to predict exposure to BaP or other carcinogenic PAHs becausethe relative concentrations of pyrene and BaP may vary widely (Srogi, 2007).

It should be noted thatthe concentration or excretion of parent PAHs compounds or metabolites in bodyfluids or urine is not only dependent on external exposure but also on theabsorption, biotransformation, and excretion, which can be significantlydifferent among individuals. BaP-DNA adduct in peripheral lymphocytes and withproteins such as albumin have also been used as indicators of reactivemetabolites. As binding of electrophilic PAHs metabolites to DNA is thought tobe a key step in the initiation of cancer, measurement of DNA adducts could bean indicator of PAHs exposure and also of the dose of the ultimate reactivemetabolite (Perera et al., 2011). Approachesof PAHs metabolomics studyMassspectrometry is widely used as a metabolomics analysis platform because itprovides high sensitivity, reproducibility, and versatility. It measuresmolecules and the mass of fragments to confirm their identity. This informationis gained by measuring the mass?to?charge ratio (m/z) of ions that are formedby inducing the loss or gain of a charge from a neutral species. A complexmixture of a sample containing metabolites can be directly measured using aseparation method such as liquid chromatography and gas chromatography and thenanalyzed 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 metabolomicsexperiments, before entering the mass spectrometer,it is recommended to usechromatographic separation to minimize signal suppression and allowfor greater sensitivity. In addition, use of the retention time can furtherhelp metabolite identification. In addition to the m/z and the retention timeinformation, ion recognition is promoted by the fracture pattern, which can beobtained through tandem mass spectrometry (Johnson et al.

, 2016).The study by Wang et al.,(2015) used an LC-MS metabolomics methodcombined with multivariate statistical data analysis to investigate human bodymetabolic disturbances after PAHs exposure. This was achieved by analyzing theurine samples of a large population of children and elderly people living in anarea polluted by the coking industry anda non-polluted control area. Metabolic alterations in response to PAHs exposurewere evaluated to discover potential metabolic biomarkers. In addition, a sensitiveliquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used formeasuring nine urine metabolites of PAHs to assess the level of exposure to thesame group of PAHs.

Finally, the individual PAHs exposure and its metabolicconsequences were assessed by correlation analysis to determine dose-effectrelationships. 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 PAHsexposures. Figure 4. Long-term PAHsexposure environment of general population research by metabolomics methodbased on LC-MS (Wang et al.

, 2015). Dataanalysis of PAHs metabolomics studyBecause metabolomics generate large data sets, computational toolsfor processing and interpretation are very important. Since big data processing,statistical analysis, metabolite identification and biological explanationrelated problem are not trivial, there are now tools (e.g., automation) thataccelerate computational workflows and provides a user-friendly tool for bothbeginners and professional scientists. The development of chemical informationtools, which are used for the calculation of metabolomics results caneffectively support the experimental data upload, processing, statisticalanalysis and identification of metabolites, and when combined withbioinformatics tools, can put metabolites in a biological environment.

Metabolomics analysis, especially not-targeted metabolomics, can lead to verycomplicated data sets. They containinformation on thousands of ions that are generated in the mass spectrometerfrom each sample, in which the ions represent the precursor intact metaboliteor its fragments, adducts or isotopes. Therefore, applyingcomputing tools to reduce the redundancy of these complex datasets and to identify the most relevantmetabolites is very important (Wolf et al., 2010; Johnson et al., 2015, 2016).Multivariate techniquesare most widely used in big data analyses of PAHs metabolomics studies such as principalcomponents analysis (PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal PLS-DA (OPLS-DA).

In addition, volcano plots can beuseful to show fold change versus significance as p-value, and heat maps canshow 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 ?Mof benzaanthracene (BAA) and benzaanthracene-7,12-dione (BAQ) on zebrafishmetabolic function.

Through the integration of multivariate, single variableand pathway analysis, a total of 63 metabolites were significantly changedafter 5 days of exposure. Obvious disturbance shows that BAA and BAQ affectprotein biosynthesis, mitochondrial function, neural development, vasculardevelopment, and cardiac function. As shown in Figure 5, PCA-DA and PLS-DAmodels of two-dimensional chart showed that BAA and BAQ groups and the controlgroup differ in PC1 from each other.In addition, as shown in Figure 6, the change of comparison between the controlgroup and group BAQ, metabolites increased slightly. Figure 5. PLS-DA score plot (A) and PCA-DA score plot (B) in thepositive ion mode based on the normalized data (Elie et al., 2015).

Figure 6. Heat map produced by hierarchical clustering of the mostsignificantly different metabolites obtained from the positive ion mode. Thelog 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 intensitiesand relative to a pooled average sample from the control group (Elieet al., 2015).Wang et al.

, (2015) usedthe metabolomics method based on LC-MS in order to study various levels of PAHsexposure in terms of human urinary metabolic alteration. As a result, comparedwith individuals between the exposure group and the control group, 18metabolites related to amino acids, purine and lipid metabolism significantlychanged. These findings suggested that chronic environmental exposure to lowlevels of PAHs in the human body would cause oxidative stress effects.

Inaddition, 1-hydroxyphenanthrene and dodecadienylcarnitine are potentially sensitiveand reliable biomarkers for PAHs exposure in the general population. The studydemonstrates that a metabolomic approach is a useful tool to identify thevarious metabolic changes of environmental PAHs exposure in the general populationand provides new insight into the mechanisms underlying PAHs-induced toxiceffects. The OPLS-DA scatter plot in Figure 7 showed that the exposed groupcould be clearly separated from the control group based on the 1400 peaksdetected by LC-MS. Figure 7. OPLS-DA scoreplots based on the (A) UHPLC?(+)ESI?MS and (B) UHPLC?(?)ESI?MS data fromelderly non-smokers. Blue dots, elderlynonsmokers in the control group (n = 96); red dots, elderly nonsmokers in theexposed group (n = 142) (Wang et al.

, 2015).Conclusion We are living in anenvironment in which PAHs and their derivatives are ubiquitous. Due to theirtoxic, carcinogenic, and mutagenic nature, it is significant to study theirbiological effects and corresponding mechanisms in humans. As discussed in thisreview, metabolomics analysis approach is a powerful tool for achieving thesegoals, especially by utilizing zebrafish model in the aquatic system.

The researchresults which presented in this review demonstrate the utility of LC-MS based metabolomics in combination with thedevelopmental zebrafish model to provide deeper mechanistic insights into thelink between chemical exposure and the profound impact of organisms. Applyingthis approach to more PAHs and diverse PAHs can greatly extend the metabolomicsused to predict the structure-activity relationships and potential hazards ofvarious and ubiquitous contaminants. Moreover, by applying various big dataanalysis methods, people can gain a better understanding of how PAHs and theirderivatives alter biochemical pathways to cause adverse health effects. These allwill provide significant information for toxicology and molecular biologyresearch.

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