Mitochondrial lipid peroxidation and microsomal drug-metabolizing enzyme activity of rat hepatotoxicity under heavy metals from slag waste exposure

doi:10.21203/rs.3.rs-2217628/v1

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Mitochondrial lipid peroxidation and microsomal drug-metabolizing enzyme activity of rat hepatotoxicity under heavy metals from slag waste exposure

https://doi.org/10.21203/rs.3.rs-2217628/v1

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Heavy metals from slag waste (HMSWs) have been a long-term concern around the world, posing serious environmental and visceral toxicity, especially hepatotoxicity. Investigating the leaching behavior and hepatotoxicity of HMSWs is of great significance. Based on the toxicogenomic analysis, the targets and related signaling pathways of heavy metal-induced liver injury were predicted, and the effects of different HMSWs on mitochondrial lipid peroxidation injury and microsomal drug-metabolizing enzyme activities in rats were experimentally explored. The results showed that different heavy metals might have the function of interfering with physiological processes such as oxidative stress, cell death, and energy metabolism regulation in vivo, and participate in the regulation of HIF-1 signaling pathway, peroxisomes, drug metabolism-cytochrome P450, ferroptosis, and other signaling pathways. HMSWs exposure caused weight loss, and significantly increased lactate dehydrogenase (LDH), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST) in different groups of rat liver, suggesting the presence of mitochondrial lipid peroxidation damage. Meanwhile, the activities of superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) in the liver were significantly decreased, and microsomal Cytochrome P450 1A2 (CYP1A2) and 3A1 (CYP3A1) enzyme activities were induced, predicting the involvement of the microsomal drug-metabolizing enzymes. These results indicated that metabolic interplay between microsomes and other subcellular organelles including mitochondria and peroxisome could be involved in heavy metals-induced liver injury.

Heavy metals

Hepatotoxicity

Mitochondria

Microsomes

Pathway analysis

Lipid peroxidation

In recent years, there has been a significant increase in the usage of heavy metals in the world. Heavy metals have an excellent recycling performance, and it is an important raw material for heavy metals refining to directly utilize solid slag waste, which has brought many serious environmental problems and health risks to the world. The treatment of such heavy metals from slag waste (HMSWs) requires high technical means, and improper disposal will bring major hidden dangers to environmental protection and public health [1–3]. For example, tailings and waste rock are submerged in water all year round, and various metal sulfides are quickly oxidized after contacting with air to form acid wastewater, resulting in changes in the chemical composition and physical state of minerals, and a large amount of the toxic and harmful heavy metal elements are released into the environment by the leaching, which in turn have toxic effects on human health [4–5]. Therefore it was particularly important to accurately identify its properties by studying the mechanisms of leaching toxicity including inorganic elements and compounds, such as iron and copper.

It is well known that iron, copper, cobalt, nickel, and manganese are important trace elements in the human body that play an important role in biological processes at very low concentrations and, worryingly, heavy metals are rarely excreted from the body and accumulate in various tissues, especially the liver, when their body concentrations exceed a certain threshold, producing certain toxic effects [6–8]. Heavy metals catalyze the oxidative stress response and induce the production of reactive oxygen species (ROS) in cellular mitochondria, which in turn leads to different metabolic injuries and adverse changes, such as liver injury and kidney injury [9]. Among those organs, the liver is one of the vulnerable target organs since its microvasculature allows ready access to hepatocytes and all of the blood including the absorption of heavy metals in the stomach and intestine must pass through the liver, and consequently, the liver occurs to injury [10–13]. It is important to monitor the key harmful outcomes of liver damage to ensure liver function.

Though the liver played a critical role in heavy metal metabolism, how elucidating the key events of metabolic transformation and liver dysfunction of heavy metal had not been achieved. Current studies mainly focused on single heavy metal or mixed exposure, and unfortunately, only limited little information was available on the toxic effects caused by leaching solutions from solid waste exposure. Accordingly, based on toxicogenomic analysis, this study aimed to evaluate the effects of different heavy metal leaching solutions from slag waste on oxidative stress and microsomal metabolism in rats and to further analyze the harmful outcome pathways.

Experimental materials

Copper concentrate slag, iron ore slag, copper and sulfur ore slag, and cobalt-nickel-manganese waste residue were all provided by Hangzhou Customs, China. Chromatographic grade methanol and Rhodamine-123 were purchased from Sigma-Aldrich (St. Louis, MO). Phenacetin (purity > 98%), testosterone (purity > 98%), and nicotinamide adenine dinucleotide phosphate (NADPH, purity > 98%) were procured from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Detection kits of lactate dehydrogenase (LDH), superoxide dismutase (SOD), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malonaldehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All chemicals used were of high-performance liquid chromatography (HPLC) grade and were used as received without any further purification and were obtained from Sigma-Aldrich. All solutions were prepared with deionized water. Deionized water was prepared by a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Animals And Treatments

Adult male SD rats (Shanghai SLAC Laboratory Animal Co., Ltd. license number: SCXK (Shanghai) 2017-0005), were housed in sterilized cages and fed autoclaved food and water ad libitum, under the conditions of temperature 23 ± 1°C, humidity 50%-70%, 12 h alternating light and dark. And then they were randomly divided into groups, namely the control group (NK,1.1 µM acetic acid, orally), and the groups of HMSWs exposure including the group of copper and sulfur ore slag (Cu&S), the group of copper concentrate slag (Cu), the group of iron ore slag, and the group of cobalt nickel manganese waste residue (CoNiMn). In the groups of HMSWs exposure, the rats were exposed to the saturated acetic acid solutions of HMSWs orally, and the gavage volume was V(ml) = body weight (g)×10 (ml/kg). All treatments were carried out for 4 weeks. Animal experimentation was approved by the China Jiliang University of Animal Ethics Committee and conducted in compliance with the ethical standards required by law and the guidelines for the use of experimental animals in China.

According to the national environmental protection standard HJ557-2010, different HMSWs were dried, ground into powder, and crossed 120 mesh sieves. Then 100 g were weighed and dissolved in 1.1 µM acetic acid solution. The saturated acetic acid solutions of HMSWs were prepared by stirring in the drug dissolution apparatus at 35°C for 72 h. After centrifugation (10 min at 3000 rpm), the supernatant was passed through 0.22 µm microporous filters to sterilize for animal exposure orally. By using atomic absorption spectrometer iCE 3000 (Thermo Fisher Scientific, USA), the copper concentration from copper concentrate slag solution was 2335 mg/L, the copper concentration from copper and sulfur ore slag solution was 0.41mg/L, and the iron concentration from iron ore slag solution was 0.95 mg/L. In the solution of the cobalt-nickel-manganese waste residue, the cobalt concentration was 0.44 mg/L, the nickel concentration was 0.53 mg/L, and the manganese concentration was 0.94 mg/L. A standard solution with a known concentration of each metal was used during the measuring process to verify the accuracy of the measurement.

Finally, the rats were anesthetized with mild sevoflurane and euthanized. The serum was centrifuged at 3000 rpm for 10 min, and stored at -20°C for further biochemical analysis. The liver tissues were put into sterile cryopreservation tubes at -80°C.

Toxicogenomic Analysis

To obtain the differentially expressed genes from the Comparative Toxicogenomics Database (CTD), CTD was employed to define the set of genes whose expression is influenced by Fe, Cu, Co, Ni, and Mn in the liver. The association information between chemical exposure, gene products, and diseases was analyzed to generate hypotheses about the molecular mechanisms of different heavy metals-induced liver injury in support of experimental evidence. We screened the studies irrelevant to different heavy metals toxicity, assay development, in vitro enzyme activity tests, characterization of protein structure or function, and exposure characterization. The final set related to different heavy metals-induced hepatotoxicity and genes whose expression or protein activity was increased/decreased/affected by Fe, Cu, Co, Ni, and Mn under the function model of chemical–gene interaction. By using the R package clusterProfiler 4.0 toolkit [14], the differential gene sets were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To compare gene clusters' functional profiles from the above gene sets, GO and KEGG class enrichment was significant enrichment.

Liver Biochemical Estimation

To interpret the effect of heavy metals accumulation on liver biochemical abnormalities, levels of serum AST, ALT, and LDH were measured by an automatic analyzer (SPECTRA MAX 190, Molecular Devices) as indicators of liver injury according to the kit manufacturer's instructions. They were expressed as U/L of serum.

Simultaneously, 1.0 g of each rat liver tissue from each treatment group was taken in ice-cold saline, made into 10% tissue homogenate, centrifuged at 3500 rpm for 10 min, and the supernatant was used for measuring superoxide dismutase (SOD) and malondialdehyde (MDA) levels by using the detection kits according to the manufacturer’s protocols, and using them as markers of lipid peroxidation.

Measurement Of The Mitochondrial Membrane Potential

To Investigate the mechanisms of mitochondrial damage in rat liver induced by HMSW, the membrane potential of mitochondria (Mψ) was quantified using the fluorescent dye Rh123 as a marker of mitochondrial membrane potential. Taking 1g of each rat liver tissue was cut up in 35 ml of pre-chilled isolation solution (225 mM mannitol, 75 mM sucrose, 0.05 mM EDTA, 10 mM Tris-HCL, pH 7.4), homogenized intermittently with an ice bath homogenizer at 9500 rpm for 30 seconds three times, centrifuged subsequently at 600 rpm for 5 min, and discarded the precipitate. The obtained supernatant was transferred to a pre-cooled centrifuge tube and centrifuged at 4℃, 8000 rpm for 30 min. Then, the obtained supernatant was discarded, and the precipitate was washed three times with 4℃ separation solution. The resulting pellet t was the purified mitochondria.

The pellet of each treatment group was resuspended in the assay buffer (containing 225 mM mannitol, 70 mM sucrose, 5 mM HEPES, pH7.2) at 25℃ and incubated with 0.3 M Rh123 for 3 min, and the excitation wavelength of 505 nm and emission wavelength of 534 nm were selected. The fluorescence intensity of each group of mitochondrial suspensions was measured, where the amount of Rh123 bound to the mitochondrial matrix reflected the value of the average mitochondrial membrane potential.

Extraction Of Rat Liver Microsomes (Rlms)

After the rats in each treatment group were executed, the abdominal and thoracic cavities were opened quickly, the rats were flushed with pre-chilled saline along the hepatic portal vein, and the inferior vena cava was cut to allow the fluid to flow out smoothly and then blotted up with filter paper so that the blood was flushed out until the liver was colorless or yellowish. The liver was cut into small pieces in an ice bath at 4℃, and the appropriate amount of liver tissue was homogenized with sucrose solution about 4 times the weight of the liver at 9500 rpm. The obtained homogenate was transferred to a centrifuge tube, centrifuged subsequently at 10240 rpm at 4℃ for 15 min, taken the supernatant and added CaCl₂ solution, centrifuged subsequently at 15000 rpm at 4℃ for 15 min, discarded the supernatant, and the precipitate was mixed with Tris-HCL buffer solution and kept in reserve. The protein concentration of the prepared liver microsomes was determined according to the instruction of the BCA protein kit.

Preparation Of Standards And Quality Control Samples

Stock solutions of phenacetin and testosterone were prepared independently in methanol at 400 and 800 µM. The working solution containing phenacetin or testosterone (5.0, 10.0, 20.0, 50.0, 100.0, 200.0, and 400.0µM) was prepared by appropriately diluting the individual stock solutions with 10 mM phosphate-buffered saline (PBS) (pH 7.4) on the day of analysis. Two calibration standards were prepared using each working solution by spiking in blank RLMs (5 mg/mL) and PBS (10 mM, pH 7.4). The concentrations of the calibrators used in the assay were as follows: 0.5, 1, 5, 10, 20, and 100µg/mL. The following quality control (QC) samples were prepared in the same manner: 0.5, 5, and 50µg/mL. All stock solutions were stored at -20°C.

Incubation For Different Hmsws Metabolic Kinetics In Microsomes

Rat liver microsomes were preincubated with various concentrations of phenacetin or testosterone for 5 min at 37°C. The incubation mixtures were conducted at 37°C with the presence of 1 mM NADPH in 100 mM phosphate buffer (pH 7.4) in a water bath at 37℃ for 5 min. In general, the reaction was continued to be incubated for 20 min and terminated in a low-temperature refrigerator at -20℃. The terminated sample was thawed at 37℃, centrifuged at 15000 rpm for 10 min at 4℃, and the supernatant was passed through the activated solid phase extraction column, eluted with methanol, and the eluate was blown dry with N₂ and re-dissolved with the mobile phase, vortexed and mixed, and filtered by 0.45 µm filter membrane for HPLC analysis.

Chromatographic Conditions

The enzymatic kinetic parameters of the two probe drugs in rat liver microsomes were determined, and the chromatographic conditions of the different detector standards were established separately. The Hypersil BDS C-18 column (250 mm × 4.6 mm having 5.0 µm particle size equilibrated with a mobile phase consisting of methanol to Na₂HPO₄(10mM, pH8.25) (65:35, ν/ν)) was used. The flow rate was kept at 1.0 mL/min, and the column was set at 30℃. Eluents were supervised using a PDA detector at 254.0 nm. A satisfactory separation and peak symmetry for the prob drugs were obtained with the above chromatographic condition. The mobile phase of finasteride consisted of methanol to water (15:85, ν/ν), and the detector was adjusted at 254nm.

Statistical analysis

Statistical differences and significance levels were analyzed by one-way analysis of variance (ANOVA). The enzymatic kinetic parameters were calculated by the Lineweaver-Burk equation, and all processed data were expressed as mean ± standard deviation, with P < 0.01 indicating significant differences.

Comparison of key GO terms and KEGG pathways enrichment for each heavy metal

To indicate the crucial toxicity pathways in heavy metal-induced hepatotoxicity, we used GO enrichment to analyze differentially regulated genes in liver injury by different heavy metals. The key 10 enriched Biological Process terms were accessed in Fig. 1a, terms such as cellular response to chemical stress, response to oxidative stress, reactive oxygen species metabolic process, response to the metal ion, apoptosis, and ATP metabolic processes were significantly enriched in both heavy metal genes expression datasets. Meanwhile, the results for Molecular Function terms were relevant terms of antioxidant/oxidoreductase activity and peroxidase activity mostly such as antioxidant activity and heme binding and peroxidase activity, phosphatase binding and monooxygenase activity were significantly enriched in Fig. 1b. As shown in Fig. 1c, there were considerable Cellular Components terms that belong to the same enriched mitochondrial and microsomal terms containing peroxisome and oxidoreductase complex in Fig. 1b.

To improve our understanding of signaling regulation pathways by heavy metals, the KEGG pathway enrichment analysis was performed for differentially heavy metals. Our results showed the following pathways of enrichment such as drug metabolism, cellular processes, and human diseases in Fig. 1d. The results revealed that the hepatotoxicity of HMSWs was closely associated with toxicity pathways such as drug metabolism-cytochrome P450, cell death, peroxisome, ferroptosis, and HIF-1 signaling pathway.

Body Weights And Liver Biochemical Determinations

No rat death was observed in all experimental groups during the whole exposure. Serum AST, ALT, and LDH activity levels have been widely considered as the main of liver enzyme alteration for indicating liver damage. As shown in Fig. 2a, the body weight of rats exposed to different HMSWs leachates increased clearly in the 1st week, but compared with the control group there was no statistical difference in HMSWs exposure groups (P > 0.05), and subsequently, a significant difference in body weight gain occurred from the 2nd week, with a statistical difference (P < 0.01) between the control group and each HMSWs exposure group. Additionally, serum ALT, AST, and LDH levels were significantly increased in the 4th week (P < 0.01), indicating that abnormal liver enzyme levels could signal liver damage, and AST/ALT and ALT/LDH ratios, in particular, were lower than those of the control group (P < 0.01), of which the ALT/LDH ratio was less than 1 in the HMSWs exposure groups, suggesting that liver dysfunction by HMSWs might be related to liver ischemic injury.

Through detecting the values of MDA and SOD, SOD enzyme activity in heavy metals exposure groups had a significant decline in contrast to the control group in liver tissue (P < 0.01), but the reverse was the significantly elevated levels of MDA (P < 0.01) in heavy metals exposure (Fig. 3), demonstrating that oxidation/antioxidation imbalances occurred during heavy metals exposure.

Measurement Of Mitochondrial Membrane Potential And Correlation Assessment Of Liver Biochemical Abnormalities

In rat liver tissue homogenate, HMSWs exposed groups showed significantly lower MMP levels, in comparison to the control group (P < 0.01), demonstrating that mitochondrial damage could be induced by HMSWs, as shown in Fig. 4a. Moreover, the Pearson’s correlation test was done to correlate the AST and ALT activities, LDH and SOD activities, hepatic MMP and MDA levels in the groups. It was observed that, in all the groups, the MMP levels showed extremely strong negative correlations with LDH, MDA, ALT, and AST levels (R = -0.87, R = -0.82, R = -0.85, and R = -0.84), and an extremely strong positive correlation with SOD activity (R = 0.80) in Fig. 4b.

Seen from correlation relationships, a negative correlation was determined between MDA and SOD (R = -0.75, P < 0.001; Fig. 4a), while a positive correlation was identified between AST and ALT activities and LDH level (R = 0.95 and R = 0.98, respectively; P < 0.001), a positive correlation was identified between AST and ALT activities and MDA level (R = 0.74 and R = 0.73, respectively; P < 0.001), and a negative correlation was determined between SOD and LDH (R = -0.84, P < 0.001). There was a highly significant positive correlation (R = 0.96, P < 0.001) between AST and ALT activities (Fig. 4b). Based on the R package treeheatr 0.2.1 toolkit [15], we further discretized these biochemical indicators and made the interpretable decision tree for the assessment of liver biochemical abnormalities in Fig. 4c. A conditional judgment was also made for each biochemical indicator, which provided a visualization of key biochemical indicators, summarized the cut-off concentration values of key measured biochemical parameters in response to the different groups, and as shown from these data, there was a higher AST level (> 58.78 U/gprot) except for the control group and the groups of CoNiMn and Cu&S also exhibited MDA > 173.2 nmol/mgprot. Accordingly, the seriousness of hepatotoxicity was predicted by considering only the cut-off value of the AST and MDA variable.

Hplc Method Validation

As described above methods of “2.7”, the calibrators were detected according to the chromatographic conditions. The linear regression was performed with the mass concentration of testosterone or finasteride solution as the X-axis and the peak area as the Y-axis, and the standard curve of testosterone was Y = 28226X + 2027.6 (R² = 0.9998), and the standard curve of finasteride was Y = 39210X + 8190.6 (R² = 0.9999), indicating the goodness of fit of a linear model to data. Testosterone or finasteride was added to inactivate liver microsomes in appropriate amounts at mass concentrations of 0.5, 5, and 50µg/mL, and the recoveries and their intra-day and inter-day coefficients of variation were calculated from the peak areas in Table 1. Under the chromatographic conditions of this study, the average recovery of testosterone was more than 95.55%, and the intra-day and inter-day coefficients of variation were less than 7.81%, presenting an acceptable value for the method precision. All precision results were summarized in Table 1.

Table 1

Precision and spiked recoveries of testosterone and finasteride in rat liver microsomes determined by HPLC
	Concentration (µg/mL)	Recovery (%)	RSD (%)
	Concentration (µg/mL)	Recovery (%)	Intra-day	Inter-day
	0.5	95.55 ± 1.42	4.54	7.81
Testosterone	5	97.80 ± 0.85	3.39	7.69
	20	98.97 ± 1.07	3.22	6.55
	0.5	98.81 ± 1.86	2.62	7.41
phenacetin	5	95.70 ± 7.39	3.18	6.52
	20	98.62 ± 6.59	0.90	1.81

Effect Of Hmsws Exposure On Drug-metabolizing Enzyme Activity Of Cyp1a2 And Cyp3a1

The values of the enzymatic reaction kinetic parameters Vmax and Km for different hepatic drug enzymes were derived according to the Lineweaver-Burk Eq. 1/v = Km/Vmax*1/[S] + 1/Vmax, with Vmax being the maximum reaction rate and Km is the substrate concentration at half the maximum reaction rate. The effects of different HMSWs exposure on drug-metabolizing enzyme activity of CYP1A2 and CYP3A1 were shown in Table 2. The Km values of liver drug enzymes CYP1A2 and CYP3A1 were very significantly reduced in each HMSWs exposure group compared with the control group (P < 0.01), which meant that CYP1A2 and CYP3A1 in HMSWs exposure had higher affinity for its substrate than the control group, indicating that the metabolism of HMSWs exposure had mediated by CYP1A2 and CYP3A1 in rat liver.

Table 2

Enzymatic reaction kinetic parameters of different hepatic drug enzymes in rat liver microsomes
Groups (n = 6)	CYP1A2		CYP3A1
Groups (n = 6)	Vm µg/(min•mL)	Km µg/mL	Vm µg/(min•mL)	Km µg/mL
NK	0.65 ± 0.05	17.90 ± 1.30	0.63 ± 0.11	19.86 ± 4.55
Cu	0.50 ± 0.06	3.79 ± 0.18^##	0.27 ± 0.05	4.04 ± 1.55**
Cu&S	0.13 ± 0.01	6.38 ± 0.94^##	0.17 ± 0.05	6.40 ± 3.31**
CoNiMn	0.49 ± 0.08	7.00 ± 1.97^##	0.29 ± 0.08	5.24 ± 0.32**
Fe	0.35 ± 0.07	8.71 ± 0.40^##	0.33 ± 0.10	9.85 ± 2.35**
^##P < 0.01 and **P < 0.01 Compared with the control group

Copper concentrate slag, iron ore slag, copper and sulfur ore slag, and cobalt-nickel-manganese waste residue were the commonly imported hazardous and recyclable waste in customs, and exposure to such heavy metals from slag waste (HMSWs) was known to be toxic, mutagenic, teratogenic, and carcinogenic to human beings and diverse animals. Although the liver played a central role in the metabolism and detoxification of heavy metals [16], key events of metabolic transformation and antioxidant defense against HMSWs exposure in rats are still unclear. Herein, this study aimed to elucidate the HMSWs-induced hepatotoxicity by the hepatic lipid peroxidation and regulation of metabolic enzymes.

As is well known, toxic manifestations of these metals were attributed primarily to oxidative stress. Based on the toxicogenomic analysis in the present study, there were oxidative stress, cell death and energy metabolism regulation, and inflammatory response in Biological Processes analysis that was significantly enriched. Our results also demonstrated the importance of mitochondria and microsomes for metal metabolism in Cell Composition analysis and from Molecular Function analysis the involvement of antioxidant systems and lipid peroxidative damage as a key pathophysiological force in liver injury. According to the enrichment analysis of the key KEGG pathway, key cellular events and the chronological order among them were recognized. Oxidative stress was shown to be the key event 1(KE 1), followed by organelle hypoxia damage (KE 2), metabolism of cytochrome P450 (KE 3), and cell death (KE 4). The order was biologically plausible.

In the results of the key pathway analysis, there were other pathways associated with oxidative stress, such as HIF-1 signaling pathways and mitochondrial dysfunction pathways. Enzymes are necessary for normal cellular metabolism, including that of the liver, and degenerative changes caused by combined metal toxicity exhibited in the liver could alter the levels of many of its enzymes. Serum ALT, AST, and LDH activity are the most common indicators used to diagnose liver impairment, but their elevation does not equate to the presence of liver disease and may also result from physiologic causes such as poor diet and oxygen consumption. The DeRitis ratio [17] AST/ALT and ALT/LDH ratios are commonly used clinically to reflect the damage of hepatocytes. Due to the exposure time of this study, ALT mainly present in the hepatocyte plasma was released into the blood, making the DeRitis ratios smaller than those of the control group. As a marker of ischemic injury, LDH is known to be induced under the hypoxic condition, and so the serum ALT/LDH ratio can be a noninvasive surrogate to evaluate the involvement of intrahepatic hypoxia in acute liver injury [18]. Our results showed ALT/LDH < 1 and reduced body weight gain in the HMSWs exposure groups, which were consistent with the HIF-1 signaling pathway of KEGG enrichment, suggesting the involvement of the hypoxia signaling pathway in hepatotoxicity progression caused by heavy metals. As the intrahepatic hypoxia by HMSWs, energy metabolism was disturbed. To maintain its high energy demand, the body needed to generate sufficient ATP. The activation of energy metabolism pathways would promote mitochondrial oxidative phosphorylation and increase ATP production. Zimna et al. [19] revealed that HIF-1 participated in cell proliferation and angiogenesis by regulating oxygen homeostasis in the body and releasing a variety of angiogenic growth factors. Therefore, the imbalance of energy metabolism pathways may be the underlying mechanism of liver injury induced by HMSWs exposure.

The accumulation of large amounts of metals in the ecosystem leads to several toxicological problems in the exposed organisms [20]. Exposure to high concentrations of metals can over-induce the production of reactive oxygen species (ROS) through different metal-related reactions. For example, transition metal ions can change the valence state and they support Fenton and Haber-Weiss type reactions by generating superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide (H₂O₂), among others [21]. In the present study, heavy metals from slag waste (HMSWs) included iron, copper, manganese, nickel, and cobalt, and the elements all belonged to the transition metals. The redox-active metals (e.g., transition metals) may disrupt essential non-enzymatic and enzymatic antioxidant components and mediate excessive production of ROS [22–24]. Excessive ROS production and defective scavenging ability of the antioxidant defense system may lead to oxidative stress and high lipid peroxidation levels. Superoxide dismutase (SOD) is part of the cellular antioxidant defense system against the adverse effects of ROS. MDA is also a key indicator of oxidative stress in biological systems and is formed by ROS-induced membrane lipid peroxidation, which can make things worse by increasing cellular toxicity, accelerating oxidative cascades, and accelerating cell and tissue damage and necrosis through association with subcellular molecules such as proteins and nucleic acids [25]. In this study, the decrease in SOD activity and increase in MDA content essentially led to oxidative stress-induced cell death, and previous studies revealed oxidative stress of heavy metals, in agreement with our findings [26], suggesting that solid waste heavy metal slag had peroxidative properties that reduced the antioxidant capacity and free radical scavenging capacity of the rat organism, and disturbed in free radical metabolism, and made abnormal changes in peroxidation and antioxidation of the organism, and ultimately exacerbated oxidative damage to the liver by free radicals and lipid peroxides.

Energy metabolism, tricarboxylic acid cycle, β-oxidation of fatty acids, oxidative phosphorylation, and urea cycle are all carried out in mitochondria [27], and mitochondria play a key role in metabolic pathways and signaling networks. A study [28] found that mitochondria were involved in the implementation of apoptosis, and a series of changes closely related to mitochondria occurred during apoptosis, such as the deregulation of oxidative phosphorylation, the decrease of ATP, the production of intracellular oxygen reaction products (ROS), and the reduction of mitochondrial transmembrane potential (MMP). It had been reported that when hepatocytes lost their membrane integrity, non-specific intracellular functional enzymes such as liver enzymes ALT and AST were released into the plasma, increasing their blood levels [29–30], which was also verified in our study. Both aminotransferases (ALT and AST) exist in the cytosol and mitochondrial fractions in animal and human tissues as two isozymes (ie,.cytoso and mitochondria-type isozymes), possibly corresponding to ALT1 and ALT2, AST1 and AST2. In the liver, AST is located in the cytoplasm (20% of total activity) and then in the mitochondria (80% of total activity) [31–33]. In addition, our results showed that compared with the control group, the MMP values of liver mitochondria of rats in different HMSWs exposure groups were significantly decreased (P < 0.01), indicating an increase in the mitochondrial membrane permeability. The increase of the mitochondrial membrane permeability by HMSWs further aggravated the damage of organelles, especially mitochondria, and led to the loss of hepatocyte membrane integrity or necrosis resulting in the release of ALT and AST in high concentrations in the plasma. The current correlation graph also showed that there was an extremely strong negative correlation between MMP and each biochemical index except for SOD, implying that oxidative stress could impair mitochondrial membrane stability. Combined with the reduction of ALT/LDH ratios, the imbalance of energy metabolism by HMSWs in mitochondria caused organelle damage and hepatocellular hypoxia.

According to the decision tree model established for the assessment of biochemical abnormalities, high AST and high MDA values were found to be the key measured biochemical parameters in the seriousness of hepatotoxicity. As one of the final products of polyunsaturated fatty acids peroxidation and stable metabolite in the cells, caused by oxygen free radicals, MDA is widely used as a biomarker of oxidative stress, and a biomarker of cell membrane damage. As discussed by McGill [34], the most important physiological function of AST may be the maintenance of the NAD+/NADH ratio within cells. AST is a critical partner in the malate-aspartate shuttle, which oxidizes NADH in the cytosol and reduces NAD + in the mitochondrial matrix to facilitate glycolysis and electron transport, respectively. Under normal circumstances, AST is limited to the mitochondria (80% of total activity) of tissue cells and released only when oxidative damage causes an increase in mitochondrial membrane permeability, finally resulting in a rise in serum AST concentration. The degree of liver injury is directly proportional to the level of serum AST, so the level of serum AST could indirectly reflect the severity of the hepatotoxicity. As the hepatotoxicity progressed, the liver injury gradually worsened, and the level of AST and MDA gradually increased. Therefore, we proposed the decision tree model whether it can be used for monitoring toxicological effects on the liver with chronic heavy metal exposure, providing an early warning of liver injury.

CYP450 enzymes in the body are found mainly in the endoplasmic reticulum and mitochondria of the liver, and the structural and functional integrity of the liver determines to some extent the body's ability to metabolize drugs [35]. CYP450 contains the detoxification system, which is the first enzyme defense against exogenous substances, and the oxidation reaction catalyzed by this enzyme is the main pathway of drug metabolism in the body. During the defense process, a large number of free radical intermediates are produced. Many studies have shown that CYP450 levels reflect the presence of pollutants in the environment [36]. CYP1A2 is an important enzyme isoform in CYP450 in liver microsomes and plays an important role in drug metabolism, responsible for the phase I metabolism of nearly 8% of drugs. At the same time, the homology of the CYP1A2 gene between humans and rats is as high as 80% [37–38]. CYP3A is the main enzyme system involved in the first-pass effect of orally administered drugs and is one of the important causes of drug interactions. Among them, CYP3A1 is important in many toxicological aspects and plays an important role in various metabolic pathways such as drug metabolism, metabolism of harmful substances in the environment, and metabolism of endogenous steroid hormones [39]. As an extremely important monooxygenase, it can be induced and inhibited by various compounds. Through changes in the quantity or activity of this enzyme protein in vivo, it affects the blood concentration and metabolic processes of exogenous substances such as harmful substances in vivo or the environment, thereby affecting the safety and toxic effects of exogenous substances. The metabolic activities of CYP1A2 and CYP3A1 in my study both showed a significant increase after HMSWs exposure, to a certain extent reflecting the high expression of proteins. Analysis suggested that the CYP450 system was involved in the toxic effect of HMSWs on liver tissue. However, we still do not know the specific link between hepatic oxidative stress and CYP450 changes, and whether there are sequential or other links, which questions that need further investigation in the future.

At the same time, induction of cytochrome P450 by drugs and other exogenous substances causes an increase in liver weight and proliferation of peroxisomes, which in turn cooperate with other organelles in various physiological and pathological settings. In particular, peroxisomes functionally interact with mitochondria in fatty acid degradation and ROS detoxification [40]. In addition, peroxisomes contain a large number of enzymatic and non-enzymatic antioxidants including glutathione peroxidase 1 (GPX1), oxidized glutathione (GSSG) reductase (GSR), and SOD, which can protect organelles from oxidative damage. Among the numerous cell death pathways, ferroptosis is an iron-dependent non-apoptotic cell death caused by unrestricted lipid peroxidation, ultimately leading to irreversible plasma membrane damage, the basic process of which involves the generation of free radicals, fatty acid supply, and an increase in the special enzyme responsible for lipid peroxidation (cytochrome P450 oxidoreductase [POR]). While the number of peroxisomes is positively correlated with susceptibility to ferroptosis, Zou et al. [41] reported that peroxisomes lead to ferroptosis through plasmalogen biosynthesis for lipid peroxidation. And so far, mitochondrial morphological changes, iron overload, and consequent lipid peroxidation and inflammation have been recognized as major hallmarks of ferroptosis [42]. Our results of the toxicogenomics analysis were consistent with these findings. Therefore, the results suggested that HMSWs exposure may cause various forms of programmed cell death such as organismal ferroptosis, apoptosis and necrosis, but the specific mechanisms and targets of their effects on organismal ferroptosis remain to be determined.

In the actual environment, solid waste does not exist alone, but a variety of heavy metal pollutants exist at the same time. Their combined action might produce different toxic effects from a single heavy metal. Combined exposure could affect each other's absorption, transport, distribution, and excretion. Therefore, the management and control of the import of various solid waste metal slag is a problem that needs to be studied and solved urgently. Based on the adverse outcome pathway approach, the present results suggested that these heavy metals from slag waste caused some key events of lipid peroxidation damage and metabolic damage in liver microsomes and mitochondria, but their specific mechanistic pathways in vivo needed to be further explored.

Author Contribution Tongle Deng contributed to the study design and critical revision of the article. Haishuo MU contributed to the data collection and analysis, drafting of the article, and writing. Wangjun Wan and Rong Kuang contributed to review and edit the article. Jingwu song performed the data collection and analysis. All authors read and approved the final manuscript.

Funding This research was supported by the Scientific Research Fund of General Administration of Customs of the P.R. China (NO. 2019HK010) and the Scientific Research Fund of NMPA Key Laboratory for Animal Alternative Testing Technology of Cosmetics of National medical product administration (NO. GZYZH2020003).

Data availability The data used to support the findings of this study are available from the corresponding author upon request.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest.

Ethics Approval The animal protocols used in the current study were approved by the China Jiliang University of Animal Ethics Committee and conducted in compliance with the ethical standards required by law and the guidelines for the use of experimental animals in China.

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Mitochondrial lipid peroxidation and microsomal drug-metabolizing enzyme activity of rat hepatotoxicity under heavy metals from slag waste exposure

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Abstract

Figures

Introduction

Materials And Methods

Experimental materials

Animals And Treatments

Toxicogenomic Analysis

Liver Biochemical Estimation

Measurement Of The Mitochondrial Membrane Potential

Extraction Of Rat Liver Microsomes (Rlms)

Preparation Of Standards And Quality Control Samples

Incubation For Different Hmsws Metabolic Kinetics In Microsomes

Chromatographic Conditions

Statistical analysis

Results

Comparison of key GO terms and KEGG pathways enrichment for each heavy metal

Body Weights And Liver Biochemical Determinations

Measurement Of Mitochondrial Membrane Potential And Correlation Assessment Of Liver Biochemical Abnormalities

Hplc Method Validation

Effect Of Hmsws Exposure On Drug-metabolizing Enzyme Activity Of Cyp1a2 And Cyp3a1

Discussion

Conclusion

Declarations

References

Additional Declarations

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