3.1. Median Lethal Concentration (LC50) and safe concentration of carp
According to the experimental procedure of 2.3, the acute toxicity dose gradient pre-experiment was first carried out, and the lowest and highest doses of carp death at 24h and 96h were recorded: the highest dose of carp that did not cause death in 96 h was 5 mg/L, the lowest dose of all death in 24 h was 40 mg/L, and when the dose was 20 mg/L, the 96-hour mortality rate of carp was about 90%. At 30 mg/L, all carps died within 96 h (Data not shown). Therefore, the acute toxicity dose gradient exposure is set in the range of 5–25 mg/L.
The lethal concentration of PHE for carp are shown in Table.2. With the increase in the concentration of PHE, the mortality of carp also increased, displaying a significant dose-toxicity effect. Furthermore, the regression equations between the probability unit of carp mortality and the logarithmic concentration of PHE after 24-h (Eq. (3)) and 96-h (Eq. (4)) cultivation were respectively obtained as follows:
Y = 3.970 2X + 0.2275, r2 = 0.9388 (3)
Y = 0.479 8X + 0.30, r2 = 0.9229 (4)
(Y: Probability unit of mortality; X: logarithmic concentration)
Table 2
LC50 of Cyprinus carpio after 24-h and 96-h exposure to PHE.
Concentration/(mg·L− 1)
|
5.01
|
6.31
|
7.94
|
10.00
|
12.59
|
15.85
|
19.95
|
25.12
|
Log concentration
|
0.7
|
0.8
|
0.9
|
1.0
|
1.1
|
1.2
|
1.3
|
1.4
|
24 h mortality rate/%
|
0
|
0
|
20
|
20
|
30
|
60
|
70
|
70
|
Unit of probability
|
3.04
|
3.04
|
4.16
|
4.16
|
4.48
|
5.25
|
5.52
|
5.52
|
96 h mortality rate/%
|
10
|
20
|
20
|
30
|
40
|
70
|
90
|
100
|
Unit of probability
|
3.72
|
4.16
|
4.16
|
4.48
|
4.75
|
5.52
|
6.28
|
6.96
|
The carp's LC50 value of PHE after 24 h and 96 h were respectively determined as 15.926 mg/L (95% confidence interval: 14.675–17.282 mg/L) and 11.198 mg/L (95% confidence interval: 9.950-12.604 mg/L). According to the classification standard for acute toxicity of fish (Sobanska et al., 2018), PHE is highly toxic to carp. According to Eq. (1), the safe concentration of PHE to carp was calculated to be 1.12 mg/L.
3.2. Gene extraction efficiency
DNA amplification samples were subjected to preset denaturation temperature, cycle parameters, and final repair extension on a gradient PCR instrument. The agarose gel electrophoresis result of 5 µL of samples is shown in Fig. S3. The three genes were loaded and electrophoresed simultaneously. From left to right, they were respectively CYP1A, GST, and β-ACTIN, a commonly used internal reference gene. According to the mobility of each DNA and the indication of the marker, the length of DNA fragments in the PCR sample was between 500 bp and 550 bp. Compared with the β-ACTIN gene, the GST gene fragment was shorter and had a higher electrophoretic mobility, followed by CYP1A. The target genes were specifically amplified and showed good integrity. Therefore, the samples could be used for the semi-quantitative analysis, recovery, and sequencing for further gene comparison.
The sequencing and splicing results of CYP1A and GST genes and alignment results obtained from the gene bank (NCBI, National Center for Biotechnology Information) are shown in Table 3. The lengths of the two amplified genes (CYP1A and GST) were respectively 503 bp and 541 bp. Similarly, the amplified gene had a high similarity of greater than 99% to the target gene in the gene bank. Therefore, the entire RNA extraction, reverse transcription and PCR operations could be qualified.
Table 3
Alignment results of sequenced results of amplified target genesa.
Genes
|
Gene length/bp
|
Accession No.b
|
Gene similarity/%
|
CYP1A
|
503
|
AB048939.1
|
99.7
|
GST
|
541
|
LC071505.1
|
99.5
|
a. The splicing results and peak images of the sequencing results are shown in the supplementary information.
b. The serial number is the gene number in GeneBank (NCBI).
3.3. CYP1A mRNA expression and EROD activity after the exposure to PHE
The CYP1A mRNA expression in carp tissues are showed in Fig. 1. We examined the effect of 0 mg/L PHE in the brain and liver as negative control. Pearson correlation indicated the significant correlation between CYP1A mRNA concentration and time in both brain and liver. CYP1A mRNA expression levels in the liver showed a significant increase compared with those in the control group during 25-day exposure to PHE (Fig. 1a) and the induction trend gradually became more significant during the exposure. CYP1A mRNA levels were induced significantly by 0.50 mg/L PHE during the 25-day exposure. In the brain, CYP1A mRNA levels were induced compared with those in the control, but the induction effect was not significantly enhanced with the increase in exposure time or doping concentration (Fig. 1b).
To detect whether the change in CYP1A mRNA brought about the change of related metabolic characteristic enzymes, the enzyme activity of EROD was tested (Fig. 1.) We examined the effect of 0 mg/L PHE in the brain and liver as negative control. In the liver, the EROD activity did not change significantly in the initial stage, but all experimental groups resulted in a significant EROD induction in the liver in the 15th day and 25th day (Fig. 1c). In the brain, the activity of EROD in the early period of PHE exposure (Day 1, 5, and 15) was significantly induced compared with the control assays, (P < 0.05). However, on Day 25, the induction was not significant (Fig. 1d).
The exposure to PHE showed the induction effect on CYP1A mRNA expression in the liver and brain tissues of carp in the entire experimental period. After intraperitoneal injection of TCDD in goldfish, it was also found that the expression of CYP1A mRNA was induced in various tissues, and the induction effect was most significant in liver tissues (Lu et al., 2013).It was reported that after the exposure of medaka to pentachlorobiphenyl, CYP1B1 and CYP1C1 mRNA expressions were induced due to the acceleration of Phase I metabolic response (Zanette et al., 2009). If a pollutant entered the carp body, the organism could defend itself by stimulating the production of Phase I- and Phase II-related enzymes degrading external compounds. However, if excessive external substances existed, the body defense mechanism became destroyed.
If fish were exposed to PAHs, an aryl hydrocarbon receptor (AhR) was produced (Wang et al., 2017). AhR can be linked with PAHs to display the protein activity. AhR is a ligand-activating factor in the cytoplasm and can affect the changes in the metabolism of foreign substances and even damage enzymes (Duan and Zhao, 2013). (Oliveira et al., 2007) found that intermediate metabolites produced by the degradation of organic pollutants through bio-converting enzymes were often highly active and toxic electrophilic compounds, which could interact with DNA and cause various DNA damages. These effects could be considered as early warning markers of PAHs pollution.
In the brain and liver of carp, the activities of EROD gradually increased during the exposure to PHE. The similar phenomenon was observed in the liver of tilapia (Oreochromis niloticus). The short-term exposure to a low concentration of PHE induced EROD activities in the liver of tilapia (Oreochromis niloticus), whereas the long-term exposure to a high concentration of PHE inhibited EROD activities (Wenju et al., 2009). (Mu et al., 2012) also found that several PAHs with lower molecular weights caused the differential expressions of P450 enzymes. It was also reported that a low concentration of PHE activated EROD in young Sparus aurata, but inhibited it under high concentration (Correia et al., 2007). In the brain and liver of carp, the activity of EROD showed an obvious correlation with the expression level of CYP1A. Many compounds could induce EROD activity of fishes and the induction of EROD activity might be impeded since chemicals were competitively bound to the structure of AhR or CYP1A (Whyte et al., 2000). The liver is a key site of detoxification and an important target organ of PAHs (Triebskorn et al., 1997). EROD is the important enzyme assisting in the metabolism of toxic compounds. If pollutants enter the body, the organism starts stress reactions. The differential responses of transcription and expression of characteristic genes can be used as early warning parameters to measure the degree of environmental pollution. The change in CYP1A mRNA expression in the liver and brain of carp was the consequence of the stimulation of the signaling pathway. If the concentration of PHE was too high, it might cause damage and inhibit the gene expression and enzyme activity (Nahrgang et al., 2009).
3.4. GST mRNA expression and GST activity after the exposure to PHE
The GST mRNA expressions in carp tissues are shown in Fig. 2. In the liver, the expression of GST mRNA was always induced when carp was exposed to 0.1 mg/L PHE and the induction effect was weakened in the 25th day. However, the exposure to 0.5 and 1.0 mg /L PHE showed a significant inhibition effect on GST mRNA expression from the first day to the 25th day (Fig. 2a). In the brain, the experimental groups of different concentrations showed the significant induction in the first day and the induction effect was reduced in the 5th day. During the PHE exposure period, the expression of GST mRNA showed a significant inhibitory effect in the 15th day and this inhibition effect was slightly restored in the 25th day (Fig. 2b). Olsvik et al reported the different fluctuation in Atlantic cod exposed to PAHs and found that only the levels of 2 of the 6 GSTs were up-regulated expressed (Olsvik et al., 2010). Costa et al found no significant difference in GST mRNA expression in Nile tilapia exposed to benzo(a) pyrene (Costa et al., 2012).
In order to detect whether the change in GST mRNA level brought about the change in related characteristic metabolic enzymes, the enzyme activity of GST was tested (Fig. 2). In the liver, the GST activities of all experimental groups were significantly stimulated after induction (P < 0.05), but the induction effect was gradually weakened (Fig. 2c). There was no obvious change in the GST activity at the beginning of the experiment (from the first day to the 5th day) in the brain. However, during the PHE exposure period, the GST activities in the 15th and 25th days had a significant induction effect (P < 0.05) (Fig. 2d). The reports also found that 5 kinds of PAHs induced the GST activity in the carp liver (Lu et al., 2009). (Yin et al., 2007) also reported that the GST activity in the tissues of catfish was significantly increased after the exposure to PHE. (Nahrgang et al., 2009) reported that benzo (a) pyrene induced the GST activity in cod liver. However, Olinga et al. found that the short-term exposure to PHE inhibited GST activity in the kidney of Liza aurata (Olinga et al., 2008).
In the study, the expression of GST mRNA in carp liver and brain showed the early induction and subsequent inhibition effects, indicating that after the Phase I metabolic reactions, the biological activity of GST in the tissues of carp gradually became unbalanced during the exposure. After the exposure to 0.1 mg/L PHE, the induced GST mRNA expression in the liver, but the same phenomenon did not occur in the brain. The difference might be interpreted as follows. The radicals produced by Phase I metabolism are metabolized and the liver is a key organ involved in the metabolism and detoxification of substances. GSTs in most eukaryotic species contain multiple gene families, many of which are expressed in multiple cell types and especially in the liver, its expression accounted for 2–4% of total cytosolic enzymes (Schlenk et al., 2008). In the study, GST was significantly induced in the liver when carp were exposed to PHE for only one day may because the GST signaling pathway was highly expressed in the early exposure stage. In the later exposure period, carp were adapted to the PHE concentration, so GST activity increase was not significant. In the brain, GST activity was significantly induced only after the 15th day of PHE exposure probably. The concentration of PHE was lower than the safe concentration, so there was no induction in the early exposure period.
3.5. Correlation analysis
The correlation analysis was carried out to test the expression levels of characteristic enzymes and characteristic mRNA (Fig. 3). The correlation coefficient indicates that EROD activity in the liver showed a positive correlation with CYP1A mRNA level (r = 0.602, P < 0.01) (Fig. 3a). In the brain, the correlation between EROD activity and CYP1A mRNA level was less significant than that in the liver (r = 0.508, P < 0.01) (Fig. 3b). However, in the liver or brain, the correlation between GST activity and GST mRNA expression was relatively poor (liver, r = 0.395, P < 0.05; brain, r = 0.293, P < 0.05) (Fig. 3c and 3d).
In the study, the activity of EROD in the liver was more highly correlated with the activity of CYP1A mRNA expression. The liver is an important metabolism organ of heterogeneous organisms and blood mediates the relationship between the brain and other target organs and accelerates the reaction between the characteristic liver enzymes and its mRNA expression. Similar experimental results had been reported in previous studies(Costa et al., 2012; Nam et al., 2017). After the brain and muscle were exposed to soluble components of crude oil, the changes in AchE activity were not obvious compared to other organs (Bettim et al., 2016). In addition, the toxicity induced by CYP1A indicated the chemical exposure and preferential effects in various biological tissues (Whyte et al., 2000).
The cytochrome P450 system showed a highly correlation than the GST system in both the liver and brain. The cytochrome P450 enzyme system played an important role in the heterogeneous biotransformation in Phase I metabolism of fish and other aquatic animals. The activity of ethoxysalolin O-deethylase (EROD) and the CYP1A mRNA level seemed to be the most sensitive catalytic probe. The induction response of the cytochrome P450 system had been determined in many studies (Goksoyr, 1992; Anna et al., 2008). The GST is a representative enzyme in Phase II metabolism and play an important role of catalyzing or reducing oxidative substances in the metabolic body. In most cases, only the modest changes in total GST activity were reported (Henson and K., 2004). Similar studies reported that GSTα mRNA expression in all tissues showed no significant difference compared with CYP1A and ABC efflux transporters after the exposure to benzo(a)pyrene (Costa et al., 2012).
In addition, there was a significant decrease in GST mRNA in the brain after 15 days of PHE exposure. The higher GST activity and the lower GST mRNA expression in the 15th day might be interpreted as follows. From the first day to the 5th day, the activity of GST increased, thus increasing the clearance rate of PHE and reducing the overall mRNA response and GST synthesis. As a result, the mRNA expression level was reduced (Fig. 2). However, the same phenomenon was not observed in the liver, indicating the differences in the heterogeneous metabolism and PHE-induced enzymes between the brain and liver (Nahrgang et al., 2009).
3.6 Mechanism
The possible mechanism of PHE exposed to carp is shown in Fig. 4. When PHE entered the carp body, the characteristic enzymes of the I-phase reaction were activated to remove pollutants, which result in an increase in the expression of CYP1A gene. At the same time, the free radicals produced by the I-phase reaction are transferred to the II-phase reaction and stimulate the expression of GST and GST genes. The difference is that since the liver is an important detoxification organ and free radicals were eliminated in time, the expression of GST gene showed a trend of increasing first and then decreasing (as shown in Fig. 2,3). Due to the limited ability of GST clearance, the expression and synthesis of GST genes were reduced (Figs. 2,3).