Biomarkers of Catalase, Glutathione S-transferase, and Ethoxyresorufin-O- deethylase in Echinometra mathaei exposed to Polycyclic Aromatic Hydrocarbons in the northern Persian Gulf

doi:10.21203/rs.3.rs-4984329/v1

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Biomarkers of Catalase, Glutathione S-transferase, and Ethoxyresorufin-O- deethylase in Echinometra mathaei exposed to Polycyclic Aromatic Hydrocarbons in the northern Persian Gulf

https://doi.org/10.21203/rs.3.rs-4984329/v1

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The Persian Gulf is a semi-enclosed sea with unique ecological conditions, where the average level of oil pollution exceeds the minimum optimum level established for the global marine environment. Echinoderms are among the most significant marine phyla in this ecosystem, particularly because their bottom-dwelling lifestyle exposes them more to sediment pollution. Numerous biomarkers can effectively indicate the adverse effects of chemical pollutants in the environment. This study investigated the responses of Catalase, Glutathione S-transferase (GST), and Ethoxyresorufin-O-deethylase (EROD) enzymes in Echinometra mathaei specimens collected from the northern Persian Gulf. Sampling was conducted at several locations, including T-wharf Khark, north Khark, Nai-Band, Shirino, Owli, and Shoghab Beach of Bushehr. Enzyme levels were measured during both cold and warm seasons. The maximum and minimum Catalase activity among all stations and seasons were recorded in Shirino (1.95 µmol/min/mg protein) and Bushehr (0.98 µmol/min/mg protein), respectively. The highest and lowest levels of Glutathione-S-Transferase activity were observed at Khark T-wharf (9.30 nmol/min/mg protein) and Nai-Band (6.63 nmol/min/mg protein), respectively. Maximum EROD activity was measured at Khark T-wharf (0.04 µmol/min/mg protein), while the minimum was found in Bushehr (0.002 µmol/min/mg protein). Overall, no significant differences (P > 0.05) were observed regarding the relationship between enzyme activities and the presence of Polycyclic Aromatic Hydrocarbons (PAHs) in different seasons. These results suggest that the sea urchins in the Persian Gulf may possess a distinct immune system or have adapted to the pollution levels prevalent in this area.

Catalase

Glutathione S-transferase

EROD

Echinometra mathaei

Persian Gulf

Echinoderms are a group of marine animals that require saltwater environments (Raven and Johnson, 2002). They are primarily utilized in pharmaceutical, industrial, and aquarium trading (Bhakuni and Rawat, 2005). Significant research has been conducted on echinoderms in various seas globally, including those in Iran (Pawson, 2007; Dabbagh et al., 2012; Fatemi et al., 2015; Nateghi Shahrokni et al., 2016).

Due to their generally benthic habitats, echinoderms are more susceptible to environmental pollution (Hickman and Robers, 2003). Organic marine pollutants encompass organochlorine compounds, hydrocarbons, and organometallic compounds. Many polycyclic aromatic hydrocarbons found in coastal sediments may originate from oil spill or arise from natural sources (Hadjizadeh Zaker, 2022). The common marine pollutants in sediments include Polycyclic Aromatic Hydrocarbons¹ and polychlorinated biphenyls² (Albarano et al., 2021).

PAHs are classified as emerging contaminants, with over 100 different organic compounds identified, comprising two or more fused benzene rings. This class of compounds has been recognized for its potential toxicity to mammals and aquatic organisms, leading to over sixteen PAHs being designated as priority pollutants by the United States Environmental Protection Agency (USEPA, 1987). Liu et al. (2023), in a study of 16 marine species (including algae, benthos, and fish) in Laizhou Bay, China, reported high bioaccumulation factors for PAHs with 4–6 rings, while algae and benthonic animals exhibited a strong capacity to accumulate PAHs with 2–4 and 5–6 rings, respectively.

PAHs are hydrophobic compounds primarily resulting from incomplete combustion and pyrolysis of carbonaceous materials (Lohmann et al., 2007; Manzetti, 2013). These compounds are characterized by their toxicity, carcinogenicity, immunotoxicity, teratogenic effects, and mutagenicity (Okona-Mensah et al., 2005; Ding et al., 2007; Barathi et al., 2023; De Pinho et al.,2023). PAHs are ubiquitous in marine environments, rapidly adsorbing onto suspended materials, sediments, and biological tissues upon release (Dachs & Eisenreich, 2000; Kumar et al., 2021). In the European Union, regulations have been established recommending the use of freshwater fish for testing substances with a higher likelihood of toxicity to protect marine organisms. Additionally, crustaceans, algae, mollusks, and echinoderms are suggested for further ecotoxicological testing (ECETOC, 2007). Research has increasingly focused on the embryonic and larval stages of echinoderms, as these stages are more commonly employed in toxicological studies compared to adults (Martin Neil, 2009).

Several studies have reported that PAHs and PCBs cause harmful effects on the development of sea urchin embryos (Steevens et al., 1999; Pillai et al., 2003, Suzuki et al., 2015). Several biomarkers are commonly used to assist in the biological monitoring and protection of aquatic systems and to identify the adverse effects of xenobiotics, which can pose ecological, biological, and health risks to aquatic organisms (OSPAR, 2007; Hylland et al., 2008; Hagger et al., 2008; Hagger et al., 2009; Brooks et al., 2011; Santana et al., 2018; Styszko et al., 2023; Lee et al., 2024). Biomarkers can provide information on the sources of hydrocarbons and the extent of oil spill weathering in water, coastal sediments, and organisms (Tarasov et al., 2013; Wang et al., 2014; Peng et al., 2016; Pu et al., 2017; Lee et al., 2024).

In research conducted in the northern Persian Gulf, the amount of PAHs was measured in the sea urchin; Echinometra mathaei. The concentrations of PAHs in sediments and in the tissues of the organisms were estimated to be 12.8–81.25 and 16.7–35 μg/kg dry weight, respectively (Keshavarzifard et al., 2017).

Albarano et al. (2021) measured the short-term effects of PAHs and polychlorinated biphenyls in sediments on the sea urchin; Paracentrotus lividus. Their study suggested that exposure to pollutants in the mesocosm for two months resulted in morphological abnormalities in sea urchin embryos.

All biological systems produce reactive oxygen species¹ and other oxidants, as well as energy, during aerobic metabolism. Oxidative enzymes, including superoxide dismutase, catalase², and glutathione peroxidase³ (Di Giulio et al., 1995; Livingstone, 2001; Regoli et al., 2002; Valavanidis et al., 2006), ethoxyresorufin-O-deethylase⁴ (Nahrgang et al., 2013; Osse et al., 2018), glutathione S-transferase⁵ (Belmahi et al., 2023; Rist et al., 2023), are commonly used to detect the first signs of biological exposure to harmful compounds and serve as biomarkers for aquatic organisms in polluted environments.

Glutathione S-transferase is involved in various functions, including ecotoxicological processes (e.g., detoxification of PAHs), intracellular transport, and the biosynthesis of eicosanoids (George and Buchanan, 1990; Hayes et al., 2005; Schlenk et al., 2008; Bouzahouane et al., 2018; Bouzahouane et al., 2024).

Catalase constitutes the first line of enzymatic antioxidant defense (Abele et al., 2017). The activity of CAT has been measured in several studies as biomarker for a various contaminants (Van Der Oost et al., 2003; Arlinghaus, 2019; Melotti et al., 2023).

Similarly, the activity of ethoxyresorufin-O-deethylase⁶ of CYP1A⁷ subfamily has been widely used as a biomarker for PAHs contamination (Goksøyr and Förlin, 1992; Van Der Oost et al., 2003, Santana et al., 2018; Peng et al., 2023).

Lee et al., (2024); reported that the ecological health of the region was significantly affected by PAHs, and this change was evident in biometric indices (ST⁸), detoxification-related enzyme activities (including EROD, and GST), antioxidant-related enzyme activities (SOD⁹, CAT, GPx), oxidative damage-related indices (LPO¹⁰), neurotoxicity-related enzyme activity (AChE¹¹), and mRNA expression of heat shock protein (HSP¹²22-2). These biomarkers exhibited a strong correlation with PAH concentrations found in both sediment and the soft tissues of clams (Ruditapes philippinarum and Mactra veneriformis).

Numerous studies have highlighted effective biomarkers of polycyclic aromatic hydrocarbons in the adult stages of echinoderms, such as Asterias rubens (Oweson et al., 2008), Diadema antillarum (Bielmyer et al., 2005), Amphiura filiformis (Newton & McKenzie, 1995). Among the products of oxidative stress; molecules like O₂, O²⁺, H₂O₂, and OH⁺ are potent oxidants that can react with cellular components, potentially leading to the inactivation of enzymes such as LPOX¹, DNA damage, and ultimately cell death and apoptosis. The defense mechanisms that counteract these oxygen radicals include antioxidant enzymes such as SOD, CAT, and GPx (Padmini et al., 2008).

Activities of GST from sea urchin; Paracentrotus lividus were explored as potential biomarkers of environmental contamination. Results indicated that GST activity was inhibited by certain substances such as the water-accommodated fraction of #4 fuel-oil², while it was induced by others, notably benzo[a]pyrene. Therefore, GST emerged as a suitable biomarker for coastal biomonitoring (Cunha et al., 2005). Elevated EROD activity levels were observed in Dab (Limanda limanda) at stations near the oil spill; the relationship between these levels and contaminants was not determined (Kirby et al., 1999).

Furthermore, the findings were supported by reports of similar pollutants impacting various aquatic species, which exhibited decreased (Theron et al., 2014; Solan et al., 2022), increased (Sadauskas-Henrique et al., 2016; De Santana et al., 2021; Santana et al., 2022), or even unchanged (Bettim et al., 2016; Ibor et al., 2020) biomarker responses.

In general, biomarkers have predominantly been studied in mollusks, crustaceans, and fish. However, research focused on these biomarkers in echinoids, particularly sea urchins such as Echinometra mathaei remains limited. Thus, a comprehensive review could illuminate fundamental issues in ecotoxicological studies, including exposure routes, exposure duration, and PAH concentrations used in experimental assays.

In this study, we investigated the activities of catalase, GST, and EROD in E. mathaei as potential biomarkers of PAH pollution in the coastal zone of the Persian Gulf. Our focus will be on the change patterns of these biomarkers commonly measured in sea urchin tissue. Additionally, we hypothesize that different sea urchin species will exhibit varying biomarker responses, with benthic species expected to show a stronger response to PAHs than pelagic species. This leads us to our central question: Can biomarkers demonstrate changes when exposed to PAHs?

Intertidal sampling

A total of six sites (Fig. 1) were sampled in the northern Persian Gulf; specifically, at Shoghab Beach in Bushehr, Owli, Shirino, Nai-Band, T-wharf and the northern coast of Khark Island. Sampling took place in March (warm season) and July (cold season) of 2016. These sites were selected due to their proximity to various sources of human pollutants, including oil extraction and transportation, construction activities, passenger docks, substantial raw sewage discharge, fishing, tourism, shipyards, and methanol production factories. Notably, two high-risk stations were chosen on Khark Island, owing to ongoing oil extraction activities and the presence of oil docks.

In total, approximately 180 samples of Echinometra mathaei were collected from the six stations across the two seasons. Samples (including sediments and sea urchins) were taken from three different points within each site, ensuring random selection. After collecting the sea urchins, each test was broken, and a mixture of about 10 g of various tissues inside the test, including the coelom, Aristotle’s lantern, muscles, ambulacral podia, and intestine was placed in a vial. These samples were then frozen in liquid nitrogen at -196^ºC. Additionally, about 1000 g sediment sample were stored in a Ziploc bags and transferred to a freezer set at -80^ºC.

Measurement of PAHs in samples

The Soxhlet¹ technique was employed for the extraction of PAHs from sediment and tissue samples using an appropriate solvent through a reflux cycle. The extraction methods utilized for sediments and tissues included Soxhlet extraction, Gas chromatography/mass spectrometry², and mechanical agitation. Initially, the samples were freeze-dried for 24 hours before being passed through a 50 µ sieve and homogenized. A prepared sample of 10 g was transferred into cellulose containers. A solvent mixture of 100 mL, consisting of hexane, and dichloromethane³ in a 1:1 ratio, was gradually added to a separatory funnel. The funnel was then carefully shaken to achieve a homogeneous mixture. To this mixture, 50 µL of deuterated PAHs (Naphthalene-D8, Acenaphthene-d10, and Perylene-D12) was added before proceeding to the Soxhlet apparatus. Following the addition of the standard solution, the Soxhlet system and samples were subjected to extraction for 8 hours at 150^ºC. To dehydrate the samples, they were treated with Na₂SO₄ for 24 hours. For desulfurization, the extracts were handled by soaking them with activated copper powder for another 24 hours. Initially, the volume of the extracts was reduced using a rotary evaporator, and subsequently, their volume was adjusted to 1 mL by injecting pure solvent using a stream of nitrogen gas. The samples were then analyzed using a coupled GC-MS, liquid chromatography-mass spectrometry¹ (Zakaria et al., 2002; Regoli et al., 2011b; Masood et al., 2016). A total of 16 PAHs were detected and quantified in the sample, including Anthracene, Naphthalene, Fluorene, Acenaphthylene, Acenaphthene, Phenanthrene, Pyrene, Chrysene, Fluoranthene, Benzo(k)Fluoranthene, Indeno(1,2,3)pyrene, Dibenz(a,h)anthracene, Benzo(a)anthracene, Benzo(a)pyrene, Benzo(g,h,i)perylene, and Benzo(b)Fluoranthene.

Measurement of biomarkers

Soft tissues were stored frozen at -80^ºCuntil analysis. Once thawed, the tissues were homogenized, and a standard buffer was added. Specifically, 100 mL potassium phosphate buffer (pH 7.5) was prepared, containing sodium chloride, phenylmethylsulfonyl fluoride², and a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin, and bacitracin. The homogenized samples were then transferred to vials and centrifuged at 11,000 rpm. Following centrifugation, the lipid phase was allowed to separate slowly, and the supernatant was collected for further analysis. Total body contents, which included the coelom, Aristotle’s lantern muscles, ambulacral podia, and intestine were tested for the activities of GST, CAT, and EROD enzymes (Krahn et al., 1992).

Glutathione S-transferase activity measurement

The activity of Glutathione S-Transferase (GST) was measured using the method described by Habig et al. (1974). Freshly prepared homogenates were applied to a glutathione-agarose affinity column that had been equilibrated with a 10 mM K-phosphate buffer (pH 7.4) containing 150 mM NaCl. The column was washed with the same buffer until the absorbance difference between the buffer and the eluate reached 0.05. The enzyme was then eluted using a gradient of 5 to 10 mM GSH in 50 mM Tris/HCl (pH 9.5), and the active eluates were pooled together. GST activity was quantified by measuring the formation rate of the conjugated substrate (chlorodinitrobenzene³-glutathione⁴) at 340 nm. Final concentrations of 1 mM CNDB (Sigma 237329), and 1 mM GSH (Sigma G6529) in potassium phosphate buffer (100 mM, pH 6.5) were used the reaction. Optical density was recorded at 1-minute intervals for 3-minutes at 37^°C, with results expressed in nmol minˉ¹ mgˉ¹ protein (Cunha et al., 2005; Regoli et al., 2011a).

Catalase activity measurement

Catalase (EC 1.11.1.6) activity was determined by measuring the enzyme-mediated degradation of hydrogen peroxide (H₂O₂) at 240 nm, according to the method of Luck (1963). The reaction mixture included 4.3 μM H₂O₂ in potassium phosphate buffer (100 mM, pH 7.0). GR activity was assessed indirectly through the consumption of NADPH during the reduction of oxidized glutathione⁵. The reaction mixture contained 2 mM EDTA (Sigma E5134), 0.5 mM GSSG (Sigma G4376), and 0.1 mM NADPH (Sigma N7505) in potassium phosphate buffer (100 mM, pH 7.5). GPx activity was measured by monitoring NADPH consumption in a glutathione reductase-coupled enzyme assay. The reaction mixture included 20 mM GSH (Sigma G6529), 2 mM NADPH (Sigma N7505), 10 U mL⁻¹ GR (Sigma G3664), 6 mM H₂O₂, all in potassium phosphate buffer (100 mM, pH 7.5) with the addition of 2 mM EDTA (Sigma E5134), 1 mM DTT (Sigma D9779), and 1 mM NaN₃ (Sigma S2002). SOD activity was defined as the amount of enzyme required to inhibit the rate of reduction of cytochrome-c by 50%. Measurements were taken at 550 nm, with optical density was recorded at 30-second intervals up to 1 minute at 37^°C. Its unit of measurement was µmol minˉ¹ mgˉ¹ protein (Lyon, 1909; Regoli et al., 2011a).

EROD activity measurement

The EROD activity was assessed based on the hydrolysis of the ethoxyresorufin substrate, following the methodology described by Burke & Mayer (1974). The rate of resorufin production from the EROD-mediated deethylation of the substrate 7-ethoxyresorufin was determined fluorometrically using excitation (520 nm) and emission (590 nm) filters. Stock solutions of 7-ethoxyresorufin and resorufin were prepared by dissolving measured amounts of crystalline solid in dimethylsulphoxide⁶ or methanol free of water, and stored at 4^°C in the dark. Fresh working solutions were prepared daily through serial dilution into either DMSO or 0.1 M phosphate buffer (pH 8.0). Both resorufin and 7-ethoxyresorufin are are light-sensitive, thus requiring protection from light during storage and use. The reaction was initiated by adding NADPH and continued for 60 minutes. EROD activity was measured fluorometrically based on resorufin production, with units expressed as minˉ¹ mgˉ¹ protein (Bradford, 1976; Gagné & Blaise, 1993; Whyte & Tillitt, 1995).

Statistical analysis

Before conducting the statistical analysis, we ensured that the data followed a normal distribution and that there was heterogeneity of variances. The data exhibited a statistically normalized distribution. Differences among groups were examined using a two-way ANOVA, complemented by Duncan's post-test for multiple comparisons. Additionally, the Pearson correlation coefficient was used to assess relationships, with a significance level set at P<0/05. Data analysis was performed using SPSS version 26.0, and the results were visualized using Google Earth.

The studies demonstrated that the trend in PAH concentrations varied between the sediments and tissues of sea urchins (ng/g) at six locations: Northern Khark, T-wharf Khark, Nai-Band, Shirino, Owli, and Shaghab Beach in Bushehr. The PAHs concentrations in both sediments and sea urchin tissues (ng/g) during the sampling period are detailed in Table 1, 2 and 3. A preliminary assessment of the potential toxicity of the sediments was conducted by comparing the measured contaminant concentrations to the international sediment guidelines established by OSPAR in 2000. Notably, Northern Khark and T-wharf Khark were found to pose the highest risk to biota due to the concentration of PAHs, particularly during the warmer months.

Table 1: Concentrations of analyzed PAH compounds in sediments (ng/g dry weight) in cold and warm seasons

PAH compounds in sediments (ng/g dry weight)	Cold Season						Warm Season
PAH compounds in sediments (ng/g dry weight)	T-wharf Khark	north Khark	Shaghab Beach of Bushehr	Owli	Shirino	Nai-Band	T-wharf Khark	north Khark	Shaghab Beach of Bushehr	Owli	Shirino	Nai-Band
Naphthalene	913.7	4.5	88.2	1.5	1.2	1.3	763.3	0.3	76	2	1	65.4
Acenaphthylene	8.9	0.3	2.9	2.2	0.3	0.3	2.9	1	4.2	4	0.1	7.6
Acenaphthene	842.9	3.4	8.5	1.4	1.1	30.2	58.3	0.3	5	0.3	1.2	2.2
Fluorene	411.1	0.3	2.3	4.1	0.3	21	11.9	2.35	1	6.1	0.5	5.8
Phenanthrene	332.1	2.6	3.1	0.3	1.8	7.1	4.6	41.5	1.5	0.33	0.1	0.3
Anthracene	30.2	0.3	2.9	16.6	0.3	0.3	90.5	1	0.1	18	0.3	5
Fluoranthene	98.2	6.7	3	7	2.6	10	6.7	17.7	1	0.41	3.4	2.9
Pyrene	392.7	1	6.1	0.3	4.4	40.3	15.6	6.6	2	2.2	5	0.3
Benzo(a)anthracene	23.3	1.3	0.1	0.3	0.3	0.3	4.7	4.55	0.6	1.03	0.5	0.3
Chrysene	40.6	0.2	0.3	0.3	0.3	0.3	4.1	0.3	0.5	1	0.6	0.3
Benzo(b)Fluoranthene	39.4	0.3	0.4	0.3	0.3	0.3	10.3	0.3	0.9	1	0.5	0.3
Benzo(k)Fluoranthene	16.7	0.1	0.1	0.3	6	0.3	4.6	0.3	0.3	0.76	5.05	0.3
Benzo(a)pyrene	45.2	0.3	0.2	0.3	0.3	0.3	5.7	2.04	0.4	0.5	0.2	0.3
Indeno(1,2,3)pyrene	26.5	0.3	0.4	0.3	0.3	0.3	4.6	0.5	0.3	0.5	0.4	0.3
Dibenz(a,h)anthracene	0.3	0.3	0.14	0.3	0.3	0.3	1.8	17.3	0.3	0.4	0.1	0.3
Benzo(g,h,i)perylene	22.8	1.8	0.3	0.3	0.3	0.3	5.5	0.3	0.6	1.1	0.3	0.3

Table 2: Concentrations of analyzed PAH compounds in tissue (ng/g dry weight) in cold and warm seasons

PAH compounds in tissue (ng/g dry weight)	Cold Season						Warm Season
PAH compounds in tissue (ng/g dry weight)	T-wharf Khark	north Khark	Shaghab Beach of Bushehr	Owli	Shirino	Nai-Band	T-wharf Khark	north Khark	Shaghab Beach of Bushehr	Owli	Shirino	Nai-Band
Naphthalene	15.4	238.2	480	119.7	99.1	43.4	293.2	132.4	120.2	101.5	96.9	94.1
Acenaphthylene	11.2	28.9	46.8	10.5	14.7	25	9.6	63.8	21.7	44.9	14.8	14.4
Acenaphthene	34.6	196.4	122.7	61.5	54.7	64	111.5	67.7	40.2	29.1	39.1	43.3
Fluorene	12.7	40.4	55.5	19.6	15.1	n.d.	40.6	254.1	36.1	92.9	34.9	33.9
Phenanthrene	87.6	175.5	231.4	110.8	52.9	171.6	371.5	2047.8	55.1	149	99.8	165
Anthracene	27.1	85	n.d.	26	18.2	40.6	63.4	83.6	36.2	30.7	19	30.5
Fluoranthene	42.4	40	n.d.	32.7	44.9	33.5	85.2	117.2	17.1	15.2	25.1	47.5
Pyrene	45.4	13	88.6	31.6	55.4	42.6	61.9	57.3	97	33	31.7	172.6
Benzo(a)anthracene	20.3	3	14	12	4	4	44.1	5.7	34	35.5	20.1	29.5
Chrysene	11	1.2	9.7	8.1	2.1	1.1	15.6	5.3	13.4	11.2	14	12.1
Benzo(b)Fluoranthene	3	1.1	5	1.8	1.3	1.4	5	2	6.7	5	3.6	4
Benzo(k)Fluoranthene	2.7	1.5	1.1	1.2	1.7	1.6	5.3	2.4	5.4	6.3	3.8	3.2
Benzo(a)pyrene	2.5	2.3	2	1	1.1	1	4	3	4.6	4.7	3.6	4.5
Indeno(1,2,3)pyrene	2	2.3	104	1.9	2.5	1.8	1.9	2.1	80	3.3	2.8	3.4
Dibenz(a,h)anthracene	1.1	1.5	3	0.9	2.7	2.7	2.6	2	1.4	1	2.9	3
Benzo(g,h,i)perylene	165.1	345	59.2	147.2	134.8	160.7	138.3	224.9	162.1	99.8	162.9	213.8

n.d.: Not detected.

The highest concentration of PAH compounds was observed in sediments and tissues respectively; at T-wharf Khark, where Naphthalene reached 913.7 ng/g dry weight during the cold season, and Northern Khark, where Phenanthrene was measured at 2047.8 ng/g dry weight during the warm season.

Table 3: Comparison of PAH compositions of sediments and tissues of sea urchins (ng/g) (P<0.05)(Avg± S.E)

Sample	Station Season	T-wharf Khark	north Khark	Nai-Band	Shirino	Owli	Shaghab Beach of Bushehr
Sediment	Warm	1234.9±131^a	3059.9±1956^a	883.6±219^a	505.2±597^a	445.8±657^a	488.8±614^a
Sediment	Cold	441.6±272^a	1024.4±310^a	581.3±132^a	489.9±224^a	559.5±154^a	1188.3±474^a
Tissue	Warm	90±1^a	194±102^a	121±29^a	15.3±75^a	32±59^a	94.7±3^a
Tissue	Cold	81±4^a	119±42^a	109.9±33^a	12.1±64^a	23.9±52^a	141.1±37^a

The changes in PAH concentrations were variable in the sediments and tissues of the sea urchins across six different stations: North of Khark Island, T-wharf of Khark Island, Nai-Band, Shirino, Owli, and Shaghab Beach in Bushehr (ng/g).

The highest levels of PAH were observed in the sediment’s duration the warm season at Northern Khark, T-wharf of Khark, Nay-Band, Shirino, Bushehr, and Owli, respectively. Conversely, the highest PAH concentrations in sediments during cold season were recorded in the Bushehr, followed by Northern Khark, Nay-Band, Owli, Shirino, and T-wharf Khark.

In sea urchin tissues, the highest PAH levels during the warm season were found in Northern Khark, Nay-Band, Bushehr, T-wharf Khark, Owli, and Shirino, in that order. During the cold season, the highest concentrations in tissues were again found in Northern Khark, followed by Bushehr, Nay-Band, T-wharf Khark, Owli, and Shirino. Importantly, the differences in PAH concentrations in sediments and sea urchin tissues across the two seasons were not statistically significant (P > 0.05), as shown in Table 3.

Table 4: Correlation coefficient study between variables

	Sediment/warm	Sediment/Cold	Tissue/warm	Tissue/Cold
Sediment/warm	1
Sediment/Cold	0.346	1
Tissue/warm	0.853	0.558	1
Tissue/Cold	0.529	0.646	0/882	1

The correlation and covariance between stations, seasons, sediments and tissues did not reach a statistically significant level (P>0.05). The regression coefficient, calculated based on a positive Pearson correlation test, indicated that changes in PAHs in sediments and tissues were directly related, showing positive covariance. As the concentrations in sediments increased or decreased, corresponding changes in the tissues exhibited similar trends (Table 4).

Additionally, the activity levels of enzymes in the tissues of Echinometra mathaei were measured at the studied stations during both the cold and warm seasons.

The activity levels of the Glutathione-S-Transferase (SGT) enzyme, expressed as nmol/min/mg protein, were measured at the T-wharf Khark, north Khark, Nai-Band, Shirino, Owli, and Bushehr stations during the cold season, yielding values of 9.30, 8.07, 6.63, 7.96, 7.11, and 6.82, respectively. In the warm season, the enzyme activities were recorded as 9.20, 8.91, 8.62, 8.56, 8.07, and 7.04, respectively (Fig 2). The results indicated that GST activity was not significantly different across the various stations. Furthermore, the highest enzyme activity was observed at the T-wharf Khark station, while the lowest was found at Nai-Band across both seasons. Overall, the relationship between GST activity and available tissue PAHs did not show significance (P>0.05) in different seasons.

The activity levels of the catalase enzyme, expressed as µmol/min/mg protein, were measured at the T-wharf Khark, North Khark, Nai-Band, Shirino, Owli, and Bushehr stations during the cold season, yielding values of 1.32, 1.34, 1.35, 1.71, 1.49, and 0.98, respectively. In the warm season, the enzyme activities were recorded at 1.26, 1.02, 1.03, 1.95, 1.36, and 1.58, respectively (Fig 3). The results indicated that catalase activity was not significantly different across the various stations. Moreover, the highest and lowest enzyme activities among all stations in both seasons were found at Shirino and Shaghab Beach of Bushehr, respectively. Overall, no significant relationship was observed (P>0.05) between catalase activity and available tissue PAHs in different seasons.

The enzyme activity levels of EROD, expressed as µmol/min/mg protein, were measured at the T-wharf Khark, north Khark, Nai-Band, Shirino, Owli and Bushehr stations during the cold season, yielding values of 0.0267, 0.0023, 0.0033, 0.0030, 0.0033, and 0.0020, respectively. In the warm season, the enzyme activities were recorded as 0.0400, 0.0113, 0.0030, 0.0060, 0.0183, and 0.0233, respectively (Fig 4). The results indicated that EROD activity was not significantly different across the various stations. Additionally, the highest and lowest enzyme activities among all stations in both seasons were observed at T-wharf Khark and Bushehr Beach, respectively. Overall, no significant relationship (P>0.05) was found between EROD activity and available tissue PAHs in different seasons.

A two-way ANOVA was conducted to examine the relationship between enzyme activities and tissues of sea urchins across different seasons. The analysis revealed no significant relationship (P>0.05) between the tissues and enzyme activities in the various seasons. Overall, the results indicated a direct relationship between PAH levels in tissues and enzyme activity, characterized by a positive correlation; as the levels of PAHs increased or decreased, the corresponding enzyme activities also increased or decreased.

Nowadays, coastal cities are experiencing increasing population and pressures of environmental exploitation. About 40% of the world's population lives within 100km of the coast (Agardy & Alder, 2005). This increasing population growth in coastal areas leads to the entry of human pollutants (industrial materials, pesticides, heavy metals, and domestic wastewater) into aquatic environments (Schwarzenbach et al., 2006). Polycyclic aromatic hydrocarbons are a group of more than 100 chemicals that are formed during the incomplete combustion of coal, oil, gas, wood, or other organic materials (ATSDR, 1995). These materials are everywhere in the biosphere; These materials are present in air, water, and soil. The levels of PAHs are increasing every year in the world's oceans due to human activities (such as oil spills and fossil fuel combustion) (Woo et al., 2006).

In the present study; the highest amount of PAH accumulation (ng/g) was observed in the warm season in the northern Khark (3059.9), followed by T-wharf Khark (1234.9), and in the cold season in Bushehr Beach (1188.3), followed by north of Khark (1024.4). Also, the highest amount of PAH accumulation (ng/g) was observed in the tissues of sea urchin in the warm season, in the northern Khark (194), followed by Nai-Band (121), and the cold season, in the northern Khark (119), followed by Bushehr Beach. However, no significant difference (P>0.05) was observed in the amount of PAH accumulation in the sediments or the tissues of sea urchins between the warm and cold seasons.

Ravanbakhsh et al., (2023) calculated 13 PAH concentrations in water and sediments of eight estuaries in the northern coastline of the Persian Gulf. The range of Σ13 PAHs concentration was 0.24–8.83 µg L^−l and 3.1–11.46 µg g⁻¹ dry weight, and the mean value was 4.99 µg L^−l and 6.06 µg g⁻¹ dry weight in seawater and sediment, respectively. Similar pollution levels were observed in the sediments of two studies.

Belmahi et al., (2023) conducted a study of multi-markers in the black sea urchin, Arbacia lixula, and found that temperature plays a role in the activity of stress enzymes. Cold periods increase the activity of Glutathione S-transferase and Catalase.

Invertebrates and vertebrates cannot modify environmental physical factors such as photoperiod, temperature, salinity, humidity, oxygen content, and food availability to suit their needs. Therefore, they have evolved mechanisms to modulate their metabolic pathways and cope with changing environmental challenges for survival. Antioxidant defenses are one such biochemical mechanisms (Chainy et al., 2016).

Since 1970, environmental monitoring programs have been developed based on chemical analyses of major pollutants such as PAHs, PCBs, heavy metals, and organochlorine herbicides, in different environments (water, sediment, and soil). Biomarker studies conducted in marine organisms related to pollution have generally focused on fish (Arufe et al., 2007) and mollusks (Binelli et al., 2006). There is limited information about echinoderms phylum (Everaarts et al., 1998; Den Besten et al., 2001; Angelini et al., 2003; Pesando et al., 2003; Cunha et al., 2005). During various studies, it has been determined that one of the possible reasons for the greater sensitivity of echinoderms compared to mollusks is the difference in their contact surface area. Echinoderms have a much larger surface area than mollusks, making them more sensitive to water pollutants and allowing them to absorb more pollutants (Martin Neil, 2009).

Biomarkers are measurable biological parameters that change in response to xenobiotic exposure and other environmental physiological stressors at various levels, including biochemical, cellular, histological, physiological, and behavioral levels in biological organisms. Biomarkers can indicate the exposure of living organisms to physical or chemical contaminants and their toxic effects (Depledge & Fossi, 1994). Several biomarkers have been utilized to measure environmental pollution, including the induction of cytochrome P450 (Everaarts et al., 1998), metallothioneins (Temara et al., 1997), heat shock proteins (Oweson et al., 2008), micronuclei (Leaney, 2003), DNA strand breaks (Frenzilli et al., 2009), cellular membrane stability (Stabili & Pagliara, 2009), phagocytosis (Coteur et al., 2002), heart rate (Felten et al., 2008), tissue regeneration (Candia Carnevali et al., 2001), osmoregulation (Dissanayake et al., 2008), feeding rate (Valentincic, 1991a,b), valve gape (Canty et al., 2007), burrowing behavior (Newton & McKenzie, 1998a,b), prey localization and righting behavior (Kleitman, 1941; Axiak & Saliba, 1981), reactive oxygen species production (Coteur et al., 2001), Glutathione S-transferases (Hagger et al., 2008; Hagger et al., 2009), UDP-glucoronosyl transferases (Regoli et al., 2011b), catalase (Hagger et al., 2008; Hagger et al., 2009), glutathione peroxidases (Regoli et al., 2011b), superoxide dismutase (Regoli et al., 2011b), glutathione (Hagger et al., 2008; Hagger et al., 2009), glutathione reductase (Regoli et al., 2011b), glucose 6-phosphate dehydrogenase (Regoli et al., 2011b), and γ-glutamylcysteine ligase (Regoli et al., 2011b).

The impact of marine pollution on biomarkers in echinoderms (common starfish, Asterias rubens; purple sea urchin, Paracentrotus lividus; and common brittle starfish, Ophiothrix fragilis) has been documented (Martin Neil, 2009)

In the present study, the activity levels of enzymes (GST, CAT, and EROD) were measured in the tissues of Echinometra mathaei.

The highest and lowest catalase activities were recorded at Shirino (1.95 µmol/min/mg protein) and Bushehr (0.98 µmol/min/mg protein), respectively. Similarly, the levels of Glutathione S-transferase activity were highest at Khark T-wharf (9.30 nmol/min/mg protein) and lowest at Nai-Band (6.63 nmol/min/mg protein). The EROD enzyme activity showed the highest values in T-wharf Khark (0.04 µmol/min/mg protein) and the lowest in Bushehr (0.002 µmol/min/mg protein). Overall, no significant differences (P>0.05) were observed concerning the relationship between the activities of these enzymes and the organisms exposed to PAHs across different seasons.

Similar studies have investigated the activity of these enzymes in other aquatic animals. Several studies have demonstrated that the activity level of catalase changes due to exposure to environmental pollutants. This enzyme can be serve as a suitable biomarker for tracking the initial effects of various pollutants (Bernhoft et al., 1994). Specifically, the catalase activity in the erythrocytes of carp fish was increased when exposed to Paraquat. Although some experiments indicated an increase in hepatic catalase activity in this fish when exposed to PCBs, BKME (bleached kraft mill effluent), and PAH-containing sediments, most studies did not report significant changes (Chelikani et al., 2004).

In this study, the changes in PAH levels affected the activity of the catalase enzyme, although no significant differences were observed. Most marine organisms in their early developmental stages (e.g., eggs or embryos) are more sensitive to chemical stress than adults, representing the weakest link in an organism’s life cycle (His et al., 1999; Martínez-Gómez et al., 2020). Wafia et al., (2017) determined the effects of contaminated freshwater on oxidative biomarkers in carp eggs, Barbus callensis (Luciobarbus callensis) in Algeria by evaluating catalase activity. Their results indicated that the oxidative status of fish eggs in freshwater was significantly different based on water quality. This suggests that the antioxidant activity of fish eggs is better in fresh, non-polluted water, while exposure to polluted water disturbs antioxidant protection, leading to increased oxidative stress and affecting the reproductive process. The duration of exposure to pollution was an influencing factor on catalase activity.

Catalase activity varied in response to pollutants; in some organisms, it decreased, while in others, it increased. A decrease enzyme activity was noted in mullet, Mugil cephalus (Padmini et al., 2009), European sea bass, Dicentrarchus labrax (Maria et al., 2009), and Nile tilapia, Oreochromis niloticus (Atli et al., 2006). In contrast, enzyme activity increased in gray mullet, Mugil sp. (Rodriguez-Ariza et al., 1993), and sardine, Sardina pilchardus (Peters et al., 1994). These findings illustrate the different effects of oil pollutants on catalase activity across various organisms.

In recent years, Glutathione S-transferases (GSTs) have been proposed as biomarkers for pollutants such as PAHs and PCBs (Fitzpatrick et al., 1995). The GST enzyme plays a crucial role in eliminating oil pollutants from the body. After PAHs enter the body, they are detoxified by monooxygenase enzymes. If the conditions for excretion of these toxic substances are not met, they can be transformed into carcinogenic and toxic molecules. Increased solubility of these toxic substances facilitates their excretion through urine (Bard, 2000). The activity of the GST enzyme can be inhibited or enhanced, similar to catalase, when exposed to PCBs and PAHs.

In most fish species, exposure to PCBs and PAHs typically leads to an increase in hepatic glutathione S-transferase (GST) activity; however, a significant decrease in GST has been reported in salmon, sea bass, and sunfish (Van Der Oost et al., 2003).

The effects of dispersants on the activity of catalase, superoxide dismutase, glutathione S-transferase, and glutathione peroxide in the intestines and glands of the sea urchin, Hemicentrotus pulcherrimus showed that the activity of all four enzymes initially increased but then decreased with rising oil leakage rates. Notably, there were no significant difference (P>0.05) in the activity of these four enzymes within the control groups (Meina et al., 2015).

PAH-induced stress can stimulate GST activity (Kang et al., 2022). Studies have demonstrated varying effects of PAHs on GST activity across different organisms. For example, some fish, such as zebrafish; Danio rerio and fathead minnow; Pimephales promelas (Boehler et al., 2018), catfish; Carassius auratus (Yin et al., 2007), polar cod; Boreogadus saida (Nahrgang et al., 2009), exhibit increased GST activity in response to PAHs, while other species, such as golden grey mullet; Liza aurata (Olinga et al., 2008). show decreased enzyme activity. This variation suggests differing roles and responses of GST to PAH exposure in different organisms.

These pollutants likely induce oxidative stress, resulting in a limited increase in the activity of antioxidant enzymes such as SOD, CAT, GR¹, GPOX², and GST in sea urchin; Sterechinus neumayeri. Interestingly, sea stars; Patiriella regularis, and Odontaster validus exhibited a higher level of these enzymes compared to sea urchins. This variation may be attributed to the impact of environmental CO₂ pressure, suggesting that starfish demonstrate greater adaptability to water acidification than sea urchins (Fleury, 2016).

Regarding GST activity, significant differences were observed in samples exposed to oil for one month; however, GST activity decreased following seven months of exposure (Angelini et al., 2003). Research also indicates that oxidative stress can initially stimulate the activity of antioxidant enzymes (Zhang et al., 2004), yet prolonged oxidative stress generally leads to decreased and inhibited enzyme activity (Niu et al., 2019).

The enzyme EROD plays a critical role in metabolizing toxic compounds. When pollutants enter an organism's body, stress reactions are triggered, making EROD activity a useful early warning indicator of environmental pollution levels (Nahrgang et al., 2009).

In studies exposing fish to PAHs, EROD and GST activities significantly increased, while CAT activity decreased (Santana et al., 2018). Specifically, phenanthrene demonstrated a strong induction effect on EROD activity. Low concentrations of PAHs stimulated EROD enzyme activity in carp, whereas high concentrations inhibited it. These findings indicate that long-term exposure to low levels of phenanthrene can pose risks to the oxidative systems of carp (Kang et al., 2022).

The range of antioxidant enzyme activity varies in response to oxidative stress, often showing initial stimulation followed by inhibition. For example, EROD activity increased over time and with rising concentrations of PAHs in the liver and brain of brown trout; Salmo trutta with more pronounced induction in the liver, which is a key site for detoxification (Triebskorn et al., 1997).

Inhibition or inactivation, along with enhanced EROD activity, has been reported at high concentrations of certain contaminants, particularly heavy metals, PAHs, and some PCB congeners. Increases in EROD activity have been documented in species such as Antarctic rockcod; Notothenia coriiceps (McDonald et al., 1995), blue-striped grunt; Haemulon sciurus (Stegeman et al., 1990), common dab; Limanda limanda (Burgeot et al., 1994), eel Anguilla anguilla (Van Der Oost et al., 1996), european perch; Perca fluviatilis (Forlin & Celander, 1993). Conversely, decreased EROD activity has been observed in the European bullhead; Cottus gobio (Fent & Bucheli, 1994), common carp; Cyprinus carpio (Banca et al., 1997), brown bullhead; Ictalurus nebulosus (Gallagher & Di Giulio, 1989), burbot; Lota lota (Lockhart & Metner, 1992), European flounder; Platichthys flesus (Eggens et al., 1995).

Organisms may exhibit different responses to various doses of PAHs. For instance, short-term exposure to low concentrations of phenanthrene (PHE) induced EROD activities in the liver of tilapia (Oreochromis niloticus), while long-term exposure to high concentrations inhibited these activities (Wenju et al., 2009). Similarly, low concentrations of phenanthrene activated EROD in young gilthead seabream (Sparus aurata), whereas high concentrations resulted in inhibition (Correia et al., 2007). These findings underscore the varied effects of oil pollutants on EROD activity across different organisms.

In this study, we aim to investigate biomarkers of Catalase, Glutathione S-transferase, and EROD in Echinometra mathaei exposed to PAHs, to further contribute to the pollution control based on the source of ecological, and biological. To achieve this, we analyzed the contents of PAHs in the sediment, and soft tissue of Sea urchin, E. mathaei, in the northern Persian Gulf.

Biomarkers could be applied to mark the organism behaviors toward of contaminants, but biomarkers of sea urchin are evaluated for their weak potential use in Catalase, GST and EROD enzymes are currently the low-value biomarkers for environmental risk assessment. Therefore, they are not suitable for using in biological monitoring.

Present results show that when PAHs entered the sea urchins; Echinometra mathaei body, the antioxidant enzymes expression of CAT, GST, EROD, and genes were stimulated. The results indicated the possible lack of response of the biomarkers present in this sea urchin, which were probably caused by a different immune system or the duration of exposure to pollution in these areas.

Some chemicals may induce or inhibit the activity of GST, CAT, and EROD, this interaction was generally not a disadvantage for the use of enzymes as biomarkers. Therefore, one should try to select more suitable candidate species for biological monitoring that can have a wide range between the activity of basal and induced oxidative enzymes. So, our study emphasizes the importance of responses of some biomarkers (GST, CAT, and EROD) in affecting PAHs in sea urchin tissues.

As a result, and in response to the initial presume, echinoderm biomarkers are weak but promising tools for ERA as supplements to existing chemical measures, but this important could not be a strong reason for rejecting the accuracy of response of benthic species to pelagic species against PAH pollutions.

The results, data, and figures presented in this manuscript have not been published elsewhere, nor are they currently under consideration by any other publisher.

This project received no funding, and no financial support was obtained. The authors conducted this research at their personal expense; no external funding was provided for this study.

Ethics approval and consent to participate

have been secured where applicable.

Author Contribution

The authors declare that they have no financial interests related to this study. All the authors of this article, including Setareh Badri, Shahla Jamili, Gholamhossein Riazi, and Ali Mashinchian Moradi, contributed to its writing. They have read, understood, and complied with the "Ethical Responsibilities of Authors" statement as outlined in the Instructions for Authors.The results, data, and figures presented in this manuscript have not been published elsewhere, nor are they currently under consideration by any other publisher.This project received no funding, and no financial support was obtained. The authors conducted this research at their personal expense; no external funding was provided for this study.Ethics approval and consent to participate have been secured where applicable.The authors declare that there are no conflicts of interest relevant to the content of this article.I have read the journal's policies regarding author responsibilities and confirm that this manuscript has been submitted in accordance with those policies. All materials are owned by the authors, and no permissions are required.Setareh Badri: wrote the main manuscript textother Authors: All authors reviewed the manuscript

Acknowledgement

This research was conducted at the Iranian National Institute of Oceanography and Atmospheric Sciences. Special thanks to Nariman Asadi for his significant contribution to this project, and to Ehsan Tavassolpor, Mostafa Kamyab, and Seyed Afshin Nateghi Shahrokni for their assistance with sampling."

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PAHs
PCBs
ROS
CAT
GPx
EROD
GST
EROD
Cytochrome P450, family 1, subfamily A, polypeptide 1
shell thickness
superoxide dismutase
lipid peroxidation
acetylcholinesterase
heat shock proteins
lipidperoxidase
WAF
FALC FAT4 model, Italy
GC-MS; Model Agilent HP 6890 GC with 5973 MSD, USA
DCM
LC-MS
PMSF
CNDB
GSH
GSSG
DMSO
Glutathione reductase
Guaiacol peroxidase enzyme activity

No competing interests reported.

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Biomarkers of Catalase, Glutathione S-transferase, and Ethoxyresorufin-O- deethylase in Echinometra mathaei exposed to Polycyclic Aromatic Hydrocarbons in the northern Persian Gulf

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