Sulfonamide antibiotics are widely applied for disease treatment in humans, animals or poultry, and are also a common feed additive. However, the utilization rate of such antibiotics is very low in humans and animals, and most enter the soil environment with manure in the form of the parent compound or its metabolites. In farmland soils of the Pearl River Delta and northeast of China, sulfonamide antibiotic levels have reached 321.4 µg kg−1 and 160.18 µg kg−1, respectively, with a 100% detection rate (Li et al. 2011; An et al. 2015). Copper (Cu) is commonly added to animal feed as an additive along with sulfonamide antibiotics. Therefore, it often occurs in combination with sulfonamide antibiotics in the intestinal tract or excrement of livestock and poultry, and in turn can enter farmland soil after long-term fertilization with such manure.

It has been reported that the adsorption of antibiotics to minerals is due to binding of the antibiotics to divalent metal cations (Jia et al. 2008). The sorption mechanism to metals ions involves complexation reactions owing to the presence of many carboxyl, hydroxyl, amidogen, heterocyclic groups or electron donors in antibiotics (Zhao et al. 2018). Such complexation between antibiotics and metals may have synergistic effects on the inhibition of soil organisms (Kong et al. 2006; Zhang et al. 2012), and may change the toxicological effects of pollutants to different degrees. So far, there have been numerous studies on the ecological risks of single antibiotic or heavy metal pollutants on animals (Guo et al. 2017), plants (Xu et al. 2017) and microorganisms (Zhao et al. 2019). However, studies concerning the health risks for soil biota caused by the co-contamination of antibiotics and heavy metals are still limited.

Exposure to antibiotics or heavy metals has been shown to induce generation of reactive oxygen species (ROS) in animals (Guo et al. 2017; Kaushal et al. 2019). NADPH oxidase is the key enzyme involved in ROS generation, catalyzing the conversion of oxygen to superoxide radicals at the expense of NADPH (Sagi and Fluhr 2001). To mitigate oxidative damage caused by ROS, many antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), guaiacol peroxidase (POD) and glutathione peroxidase (GPx), act together to scavenge excessive ROS (Mittler 2002). However, when ROS generation overwhelms the capacity of the antioxidant defense processes, damage may occur to intracellular proteins by oxidizing the amino acid side chains (e.g., forming carbonylated proteins). This in turn promotes protein accumulation or denaturation via covalent cross-linking, thereby leading to a loss of protein function or enzyme activity (Stadtman and Levine 2003; Grune et al. 2005).

The objective of the present study was to determine the biochemical toxicity and potential detoxification mechanisms in earthworms (Eisenia fetida) exposed to single treatment of sulfamethazine (SMZ), Cu or their combination in soil. The results may provide a scientific basis for assessment of the health risks of the combined effects of sulfonamide antibiotics and Cu on earthworms in soil and possible application of earthworms to remediate soils polluted by antibiotics and heavy metal(s).

Materials and Methods

Soil samples were taken from the campus of Huainan Normal University, Anhui Province, China. After air drying, the soil was ground and sieved through a 2 mm mesh, and 2 kg of the sifted soil was then placed in each container. The physicochemical properties and contents of representative heavy metals in the native soil were the same as previously reported by Wang et al. (2018). A stock solution of sulfamethazine (SMZ, Sigma) was prepared by dissolving a proper amount of SMZ into 0.1 M hydrochloric acid (HCl) solution. Moreover, Cu stock was prepared by directly dissolving a certain amount of CuSO4·5H2O (Sigma) into deionized water. The stock solutions were diluted and homogeneously sprayed into the soil respectively, followed by thoroughly blending. The concentrations of added SMZ and Cu were as follows: 0 (control, Ck), 7.5 mg kg−1 SMZ, 15 mg kg−1 SMZ, 30 mg kg−1 SMZ, 100 mg kg−1 Cu, 100 mg kg−1 Cu + 7.5 mg kg−1 SMZ, 100 mg kg−1 Cu + 15 mg kg−1 SMZ, and 100 mg kg−1 Cu + 30 mg kg−1 SMZ. Triplicates were conducted for each concentration, and 24 containers were prepared for each of two independent experiments.

After 1 week of equilibration at room temperature, contents of total Cu in the control and treated soils were measured according to the method as described by Wang et al. (2010). The contents of Cu were detected as 21.50 ± 2.35 mg kg−1 in the tested soils. SMZ residues in the tested soils were measured by the method of Lertpaitoonpan et al. (2015) with minor modifications. SMZ was not detected in the control and 100 mg/kg Cu-treated group. The contents of SMZ ranged from 0.382 to 2.776 mg kg−1 in the soils treated by SMZ alone, and from 0.221 to 3.432 mg kg−1 in the combined soils of SMZ and Cu (specific data not shown).

Moreover, sixteen earthworms (Eisenia fetida) with uniform size and clitellum were selected and transferred to each container according to OECD guidelines (2004). The earthworms were incubated at 21°C and 75% relative humidity for 30 days. The humidity of the soil was maintained at 30% (w/w) by adding an appropriate amount of deionized water after weighing each pot. Additionally, 0.05 kg of cow manure was spread over the soil surface in each pot. After 30 days’ exposure, the earthworms were collected, thoroughly depurated and rinsed with deionized water, and then treated as described below.

Crude enzyme extraction was performed as described previously (Wang et al. 2018). The earthworms were homogenized on ice in an extraction buffer of 0.1 M Tris-HCl (pH 7.5) containing 0.25 M sucrose, 0.1 M EDTA, 0.2% (v/v) Triton X-100, 2 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 5 mM ascorbic acid, 1 M benzamidine, 1 µg mL−1 leupeptin and 2 µg mL−1 aprotinin. Afterwards, the homogenates were centrifuged at 12,000 g for 10 min. The protein content of the supernatant was determined according to the method of Bradford (1976). All procedures were performed at 4°C and each treatment was performed in triplicate. SOD, APX and POD isozymes were detected based on methods described in García-Limones et al. (2002) and Wang et al. (2018). CAT isozyme was determined as described by Verma and Dubey (2003). An amount of crude enzyme extracted as described above was mixed with glycerin and bromophenol blue, and then loaded onto a polyacrylamide gel consisting of 4% stacking gel and 8% resolving gel. Native polyacrylamide gel electrophoresis was performed using a high-throughput Mini-PROTEAN 3 electrophoresis system (Bio-Rad) with voltages across the running stacking gel and resolving gel set at 85 and 140 V, respectively. GPx isozymes were detected according to the methods of Lin et al. (2002). A certain amount of crude protein was mixed with 2-mercaptoethanol, glycerol and bromophenol blue, and then separated on a polyacrylamide gel (4% stacking gel and 10% resolving gel). NADPH oxidase isozymes were assayed according to Sagi and Fluhr (2001). A certain amount of each sample was prepared in Tris-HCl (pH 7.8) buffer with 0.1% (w/v) 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate (CHAPS) for 30 min at 45°C and then subjected to native polyacrylamide gel electrophoresis (4% stacking gel and 8% resolving gel). Endoproteinase (EP) isozymes were detected as described previously by Wang et al. (2018). Enzyme extracts were incubated at 37°C for 16 h and then loaded onto polyacrylamide gels (5% stacking gel and 8% resolving gel) containing 0.25% (w/v) gelatin.

Protease and glutathione S-transferases (GSTs) activities were determined as described by Gajewska and Skłodowska (2010) and Habig and Jakoby (1981), respectively, with some modifications.

Total protein extraction and western blotting analysis were performed according to Wang et al. (2012, 2018). For western blotting analysis, the total protein extract was mixed with lysis buffer (0.5 M Tris pH 6.8 containing 20% (v/v) glycerol, 20% (w/v) SDS, 0.2% (w/v) bromophenol blue and 10% (v/v) β-mercaptoethanol), then heat-denatured by boiling for 5 min, cooled and centrifuged at 12,000 g for 2 min. Proteins were separated by SDS-PAGE (5% stacking gel and 12% resolving gel) and then transferred onto polyvinylidenefluoride (PVDF) membranes (Amersham). The transferred proteins were labeled with primary (1:3000) and secondary (1:10000) antibodies prior to visualization using a SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific). The primary antibodies included anti-ubiquitin (Boster Corp.), anti-proteasome 20S β2 subunit (Enzo Life Sciences, Inc.), anti-HSP 70/HSC 70 (Sigma-Aldrich, USA), anti-CYP17A1 (Sangon Biotech Co., Ltd.) and anti-DNPH IgE (Sigma Aldrich, USA). Goat anti-rabbit or goat anti-mouse antibodies (Stressgen Corp.) were used as secondary antibodies. In addition, β-actin served as a sample loading control, labeled with primary antibody (anti-β-actin, diluted 1:2000, BM0626, Boster Corp.) and secondary antibody (goat anti-mouse, diluted 1:20,000, Stressgen Corp.). All loading samples were normalized to the β-actin level.

Statistically significant differences in the results were analyzed by one-way ANOVA followed by the t-test using the SPSS software package. Differences were considered significant at p < 0.05.

Results and Discussion

Previous studies have largely focused on single contamination by antibiotics or heavy metals and few studies have examined the underlying ecological toxicity in soil animals (e.g., earthworms) subjected to a combination of antibiotics and heavy metal(s) in soil. The present study investigated the biochemical toxicity and potential detoxification mechanisms in earthworms (Eisenia fetida) exposed to SMZ alone or in combination with Cu in soil.

The electrophoresis gel band intensities (denoting enzyme activities) indicated the total activities of isozymes. As shown in Fig. 1, under the single treatment of SMZ, the activities of SOD, CAT, GPx and NADPH oxidase increased with increasing SMZ in the soil. This suggested that increased concentrations of SMZ caused ROS overproduction in earthworms. When ROS accumulation exceeds the capacity of antioxidant defense systems, oxidatively damaged proteins are produced (e.g., carbonylated proteins) (Stadtman and Levine 2003; Grune et al. 2005). Immunoblotting showed the accumulation of carbonylated proteins at 7.5–30 mg kg−1 SMZ, indicating oxidative damage induced by SMZ in earthworms (Fig. 2d). Accumulation of these damaged proteins in cells can lead to cell death (Davies and Shringarpure 2006). Thus, elimination of oxidatively damaged proteins is an important detoxification mechanism in earthworms exposed to SMZ in soil.

Fig. 1
figure 1

Changes in isozyme patterns of SOD (A), CAT (B), APX (C), POD (D), GPx (E) and NADPH oxidase (F) enzymes in Eisenia fetida exposed to each treatment for 30 days. Ck denotes no treatment (control), whereas lowercase letters denote treatment with (a) 7.5 mg/kg SMZ; (b) 15 mg/kg SMZ; (c) 30 mg/kg SMZ; (d) 100 mg/kg Cu; (e) 100 mg/kg Cu + 7.5 mg/kg SMZ; (f) 100 mg/kg Cu + 15 mg/kg SMZ; (g) 100 mg/kg Cu + 30 mg/kg SMZ

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Fig. 2
figure 2

Western blotting of ubiquitin (A), 20S proteasome (B), Hsp70 (C), carbonylated proteins (D) and β-actin (E) in Eisenia fetida exposed to each treatment for 30 days. Ck and a–g denote the same treatments mentioned in Fig. 1

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The present study showed that treatment with increasing concentrations of SMZ alone elevated the activities of SOD, CAT and GPx isozymes above those of controls (Fig. 1a, b and e), alleviating oxidative stress in the earthworms. Moreover, to eliminate abnormal proteins (e.g., oxidatively damaged proteins) in cells, several proteolytic systems have evolved in organisms. Among them, the ubiquitin-proteasome system is reported to be a major pathway for protein degradation (Smalle and Vierstra 2004). In this pathway, target proteins are first ubiquitinated by ubiquitin and then recognized and degraded by 26S proteasome (Glickman and Ciechanover 2002; Kahana 2007). The 26S proteasome consists of a catalytic core (20S proteasome) and two 19S regulatory subunits (Smalle and Vierstra 2004). As illustrated in Fig. 2a and b, levels of ubiquitin and the 20S proteasome exhibited similar trends: both tended to increase and were significantly enhanced at 15–30 mg kg−1 SMZ. Also, after treatment with Cu alone, both were distinctly induced compared with the controls. The synchronous change of ubiquitin and 20S proteasome indicated that the ubiquitin-20S proteasome pathway was mainly responsible for eliminating oxidatively damaged proteins in earthworms exposed to SMZ in soil.

In response to stress, organisms also increase the synthesis of heat shock proteins (HSP70) to repair or degrade denatured proteins (Roberts et al. 2010; Liu et al. 2019). In this study, HSP70 levels tended to increase with increasing SMZ and were markedly enhanced compared to the control under the single treatment of SMZ (Fig. 2c). These findings revealed that HSP70 was potentially involved in repairing or eliminating the oxidatively damaged proteins, thereby relieving the oxidative damage in earthworms.

Proteases also play an important role in the degradation of oxidized proteins (Knecht et al. 2009). Previous studies have reported that the proteolytic process is initiated by the 20S proteasome, which is then joined by proteases to eliminate oxidized proteins (Book et al. 2005; Polge et al. 2009). It was obvious from Fig. 3 that protease activities showed an increasing trend and were significantly elevated at 15–30 mg kg−1 under the single treatment of SMZ in soil (p < 0.05). Similarly, in comparison to the control, significant increases were observed in the integrated densities of EP isozyme bands (representing EP activities) under the treatment of SMZ alone (Fig. 3). This suggested that both pathways co-operated to eliminate oxidatively damaged proteins and were important detoxification mechanisms in earthworms.

Fig. 3
figure 3

Responses of protease and EP isozymes in Eisenia fetida exposed to each treatment for 30 days. Ck and a–g denote the same treatments mentioned in Fig. 1

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Additionally, CYP450 and GSTs are involved in the metabolism or biotransformation of endogenous harmful substances in many organisms (Tompkins and Wallace 2007; Awali et al. 2019; Sun et al. 2019). With increasing concentrations of SMZ alone in soil, both GST activities and CYP450 production were significantly increased above those of controls (Fig. 4), suggesting the involvement of CYP450 or GSTs in the detoxification mechanisms in earthworms.

Fig. 4
figure 4

Alterations in GST activities and CYP450 proteins in Eisenia fetida exposed to each treatment for 30 days. Ck and a–g denote the same treatments mentioned in Fig. 1

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When 7.5–30 mg kg−1 SMZ was added to Cu-polluted soil, the activities of SOD or CAT showed an upward tendency in comparison to Cu treatment alone, whereas APX, POD, GPx and NADPH oxidase decreased first and then rebounded in earthworms (Fig. 1). Meanwhile, the levels of ubiquitin and the 20S proteasome initially decreased and then increased with further increases of SMZ (Fig. 2a, b). Moreover, the activities of proteases and GSTs increased with increasing SMZ (Figs. 3 and 4). However, the products of ubiquitin, the 20S proteasome, HSP70 and CYP450 as well as activities of EP decreased for the combination with 7.5 mg kg−1 SMZ and then increased with higher SMZ concentrations of 15–30 mg kg−1 in the soil (Figs. 2, 3 and 4). From the above results, it could be concluded that enhanced activities of SOD, CAT, proteases and GSTs seemed to be important in alleviating oxidative stress and damage in earthworms exposed to lower doses of SMZ in combination with Cu. At higher SMZ concentrations in the combined treatment, these enzymes appeared to work together with the ubiquitin-20S proteasome, HSP70, CYP450 and EP isozymes to mitigate oxidative stress and eliminate oxidatively damaged proteins in earthworms.

In conclusion, increasing the concentration of SMZ in soils activated SOD, CAT and GPx isozymes, suggesting ROS burst in earthworms. Excessive ROS might be responsible for the accumulation of oxidatively damaged proteins (e.g., carbonylated proteins) in earthworms exposed to SMZ alone. The accumulation of carbonylated proteins elevated the production of CYP450 and HSP70, activities of proteases, EP and GSTs, and accelerated the ubiquitin-20S proteasome pathway, improving detoxification in earthworms. Under the co-exposure of SMZ and Cu, the activities of SOD, CAT, GSTs and proteases tended to increase, whereas the activities of POD, APX, GPx and EP, production of carbonylated proteins, CYP450, HSP70, ubiquitin, and the 20S proteasome first decreased then elevated with increasing SMZ. Thus, the activated SOD, CAT, GSTs and proteases alleviated oxidative stress and damage in earthworms exposed to low doses of SMZ in the combined treatments, and then these enzymes worked together with the ubiquitin-20S proteasome, HSP70, CYP450 and EP isozymes to mitigate oxidative stress and eliminate oxidatively damaged proteins in earthworms exposed to higher SMZ concentrations in the combination.

Therefore, the ubiquitin-20S proteasome, proteases, HSP70, GSTs, CYP450 and antioxidant enzymes (e.g., SOD, CAT and GPx) contribute to defense and detoxification systems in earthworms exposed to SMZ, Cu or their combination in soil.