The triggered antioxidant response and corresponding metabolomics expression of Caenorhabditis elegans for chronic exposure to moxifloxacin and trace copper

TL;DR

This study examines how moxifloxacin and trace copper affect the health and antioxidant responses of C. elegans over time.

Contribution

The study reveals new insights into the combined toxic effects of moxifloxacin and trace copper on C. elegans at multiple biological levels.

Findings

Prolonged exposure to moxifloxacin inhibits genes related to cellular health and antioxidant defense in C. elegans.

Chronic exposure to moxifloxacin and copper reduces ROS levels and promotes antioxidant defenses through gene regulation.

The study identifies specific gene and metabolic responses to chronic exposure of moxifloxacin and trace copper in C. elegans.

Abstract

As the terminal management for evaluating the engineering effectiveness of antibiotics production and utilization, the toxic effects of moxifloxacin (MOX) and trace concentration of Cu2+ (MOX-Cu) on Caenorhabditis elegans (C. elegans) were investigated at physiological, biochemical, and molecular level. Although the stimulate effects were observed after prolonged exposure (72 h) to MOX (0.2-2.0 mg/L), the expressions of HSPs, ace genes, and daf-16 were inhibited, indicating its adverse impact on cellular health, locomotion behaviors, and antioxidant defense of C. elegans. Similarly, the down-regulation of oxidative stress (sod-1 and daf-16) and cell damage (HSPs) related genes and the up-regulation of apoptosis-related genes (cep-1 and ape-1) indicated the oxidative stress and genotoxicity after prolonged exposure to MOX-Cu. For the chronic exposure (10 days) to MOX, the level of ROS was reduced due to the increased expressions of daf-16, sod-3, and hsp-16, accompanied with and the down-regulation of cep-1. Meanwhile, at the exposure to MOX-Cu, the levels of ROS and lipofuscin were decreased due to the up-regulation of sod-1 and daf-16, and the antioxidant defense was promoted and confirmed by the increase of amino acids and their related metabolic pathways. These results can provide a theoretical basis for the toxicity evaluation of typical antibiotics (MOX) that co-existing with trace heavy metals in natural environment media and bioresources processes.

The online version contains supplementary material available at 10.1186/s40643-026-01033-4.

Figures

Effects of MOX, Cu, and MOX-Cu on locomotion behaviors of *C. elegans* after prolonged exposure. A Head thrashes. B Body bends. C Body length. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups (p* < 0.05, *p* < 0.01)

Effects of MOX, Cu, and MOX-Cu on locomotion behaviors of *C. elegans* after chronic exposure. A Head thrashes. B Body bends. C Body length. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups (p* < 0.05, *p* < 0.01)

The oxidative stress of *C. elegans* after prolonged exposure to MOX, Cu, and MOX-Cu. A Representative images about ROS production. B ROS production. C Representative images about lipofuscin accumulation. D Lipofuscin accumulation. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups (p* < 0.05, *p* < 0.01)

The oxidative stress of *C. elegans* after chronic exposure to MOX, Cu, and MOX-Cu. A Representative images about ROS production. B ROS production. C Representative images about lipofuscin accumulation. D Lipofuscin accumulation. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups. (p* < 0.05, *p* < 0.01)

Expression of stress-related genes in *C. elegans* after prolonged and chronic exposure to MOX (A) and MOX-Cu (B). Values of stress-related gene expression were normalized using actin mRNA and represent means (*n* = 3) relative to the control

Principal component analysis (PCA) score plot of *C. elegans* exposed to MOX-Cu. A Positive ion. B Negative ion

Pathway analysis of *C. elegans* between the control and MOX exposure group (A), Cu exposure group (B), and MOX-Cu exposure group (C)

Affected metabolites of *C. elegans* after exposure to MOX-Cu

Full text

Introduction

As the typical emerging biological contaminant, antibiotics have been extensively used in medicine, agriculture, and the pharmaceutical industry, thus have raised global concerns and posed a significant threat to public health and water safety, due to the emergence of antimicrobial resistance and environmental contamination (Qi et al. 2025). The usage of moxifloxacin (MOX) continues to grow around the world, especially since the outbreak of global pandemic-causing coronavirus disease 2019 (Liu et al. 2021). MOX have been detected in wastewater (17–971 ng/L), municipal sewage sludge (0.2–85 mg/kg), surface water (7.2–250 ng/L), river sediment (0.87–2.8 µg/kg), and soil (0–37 µg/kg) with relatively high concentrations (Maia et al. 2020; Chen et al. 2021). Wastewater discharge, sewage irrigation, and agricultural use of sewage sludge may result in the accumulation of MOX in surface water, sediment, and soil, which will further generate significant ecological risk due to the developing of antibiotic-resistant bacteria and genes (Wan et al. 2022).

The global challenge of antibiotic contamination calls for comprehensive approaches of effective and sustainable solutions (Fatima et al. 2025). From an engineering perspective, antibiotic contamination poses threat to wastewater treatment systems and other biological treatment processes, where complex mixtures of emerging contaminants may influence system stability and treatment efficiency. The toxicity studies of MOX mostly focus on aquatic organisms. It has been reported that the growth of Microcystis aeruginosa will be inhibited by 50 µg/L of MOX (Wan et al. 2021). The median lethal concentrations (LC_50_) of MOX for Daphnia magna and Ceriodaphnia dubia was in the range of 14–73 mg/L and 3–23 mg/L after 48- and 72-hours exposure, respectively, and the median effective concentration (EC_50_) ranged from 4 to 28 mg/L after 21 days exposure (Kergaravat et al. 2021). However, the sublethal toxicity effect of MOX on unionid mussel Lampsilis siliquoidea is 120 mg/L (Gilroy et al. 2014). To date, few studies have paid attention to the toxic effects of MOX on nematodes, which are the most abundant meiobenthic and metazoan in freshwater sediment and soil, respectively (Höss et al. 2022).

Caenorhabditis elegans (C. elegans) has been successfully used for the potential ecological risk investigation of complex environmental media (soil and freshwater sediment) and waste liquid (landfill leachate) that containing various contaminants (Zhu et al. 2023). However, studies on the toxic effects of combined pollution media on C. elegans usually treat all contaminants as an integral whole, and ignore the effects of interaction between pollutants on toxic actions. The coexistence of different pollutants, including multiple pollutants of the same type or different types will significantly affect the response of nematodes (Gutiérrez et al. 2016). For instance, the pseudo toxicity abatement effect of norfloxacin-Cu combined exposure on C. elegans has been found in previous research (Liu et al. 2022).

Emerging organic contaminants including antibiotics have been detected globally in community drinking water sources and tap-water that contain extremely low concentrations of heavy metals (Mukhopadhyay et al. 2022), which are also essential trace elements for human body (e.g. Cu). Meanwhile, apart from the ubiquity of heavy metals of anthropogenic origin, the coexistence of antibiotics and heavy metals can be also found in feed additives and excrements of livestock. In such circumstance, the suitability of C. elegans for toxic identification of MOX and MOX-Cu had been investigated in this study. Consequently, the coexistence of MOX and Cu^2+^ is prevalent in bioresources processes, as well as in the products and wastewater. The identification of the combined toxic effects of MOX and Cu^2+^ therefore constitutes a fundamental scientific issue for evaluating the safety and sustainability of bioresource utilization and biological treatment processes.

Previous studies on the toxicity of MOX in C. elegans have mainly focused on short-term and single-substance exposure (Yilmaz et al. 2019). However, the toxic effects of MOX under environmental level exposure, as well as the associated regulatory mechanisms at multiple biological levels, remain insufficiently understood. It is necessary to conduct the multi-level assessment to clarify the systemic toxicity of MOX. Therefore, the suitability of C. elegans for the toxicological identification of MOX alone and in combination with Cu^2+^ was investigated in this study. The experiments were conducted with different MOX concentrations and the fixed trace concentration of Cu^2+^, and the duration time were 72 h and 10 days for prolonged and chronic exposure experiments, respectively. The changes in physiological and biochemical indicators were evaluated, and their regulation mechanisms were further investigated at the genetic level. In addition, the metabolic profiles of C. elegans were performed to elucidate antioxidant response mechanisms. In brief, the results provided mechanistic insights into stress responses induced by MOX-Cu co-exposure and generated essential data for the toxicological evaluation of antibiotic-metal combined pollution. These findings offered a scientific basis for understanding the potential biotoxicity of such combined contaminants within the application and remediation processes of antibiotics, thereby supported the optimization of bioprocesses and the safety assessment of bioresources production and application.

Materials and methods

Chemical and reagents

MOX (CAS: 151096-09-2, C_21_H_24_FN_3_O_4_, 98%) was purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. (Shanghai, China). Copper sulfate pentahydrate (CAS: 7758-99-8, CuSO_4_·5(H_2_O), > 98%) was purchased from Titan Technology Co., Ltd., Shanghai, China. TRIzol reagent and Fastking RT kit (with gDNase) were purchased from Tiangen Biotech Co. Ltd., Beijing, China. SYBR^®^ Green PCR kit was purchased from Roche Diagnostics GmbH (Indianapolis, IN, USA). All reagents used in this study were of analytical grade unless stated otherwise. Ultrapure water that prepared by Milli-Q water (Classic DI, ELGA, Marlow, UK) was used in the preparation of aqueous solutions.

Nematode strains and culture conditions

C. elegans (wild-type N2) were obtained from Caenorhabditis Genetics Center (CGC) and cultured on sterilized growth medium plates (NGM: 3.0 g/L NaCl, 2.5 g/L peptone, 17.0 g/L agar, 7.75 g/L potassium phosphate, 0.11 g/L CaCl_2_·2H_2_O, 0.24 g/L MgSO_4_·7H_2_O, and 1 mL cholesterol) at 20 °C. Meanwhile, the growth medium plates were seeded with the bacterial lawn of Escherichia coli (E. coli) strain OP50 as food. The bleaching buffer (18.0 g/L NaOH, 2% HClO) was used for the treatment of gravid hermaphrodites achieved synchronization of the nematode (Stiernagle 2006), and the synchronized populations of L1 larval stage were acquired and washed with K solution (contain 2.98 g/L of NaCl and 2.39 g/L of KCl) three times before the exposure experiments (Wang et al. 2020).

Experimental procedure

MOX was dissolved in K solution to prepare the desired stock solutions, and diluted with K solution to prepare working solution before usage. 10 mL of MOX (0, 0.2, 2, 20, 200 mg/L), Cu^2+^ (0.0001 mg/L), or MOX-Cu (0.2 + 0.0001, 2 + 0.0001, 20 + 0.0001, 200 + 0.0001 mg/L) solutions were added to 6-Well Plate, followed by the addition of synchronized L1 larvae. The exposure duration was 72 h (prolonged exposure) and 10 days (chronic exposure), respectively (Liu et al. 2022), allowing the differentiation of immediate toxic effects from long-term adaptive responses across different biological time scales of C. elegans.

Examination of physiological indexes of C. elegans

For the counting of head thrashes, the nematodes were picked onto fresh NGM plates without food, and the head thrashes that defined as the change in direction of bending at the middle body were counted for 20 s after recovery (1 min). The nematodes would be transferred onto another NGM plate (without food) to assay body bends, which would be also counted for 20 s. A change in the direction of nematodes corresponding to the posterior bulb of the pharynx along the y axis was regarded as once body bend, assuming that the nematode was traveling along the x axis (Zhou et al. 2016). For the measurement of body length, the test nematodes were killed by heat and observed by microscope (Nikon Eclipse 80i) equipped with a graduated eyepiece. Subsequently, the gathered data were analyzed by the Image J software. The experiments were independently repeated three times with sample size of 40 worms in each group.

Examination of oxidative stress and lipofuscin accumulation of C. elegans

After prolonged and chronic exposure to MOX, C. elegans were pre-incubated in different reagents. To detect ROS production, the nematodes were pre-incubated in 0.5 mg/L of 5′,6′-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate (CM-H_2_DCFDA) for 2 h at 20 °C, and then anesthetized with 60 µg/mL levamisole and transferred onto 2% agar pads. The fluorescent images were photographed by fluorescence microscope (excitation wavelength was 488 nm, emission wavelength was 510 nm). For the assessment of lipofuscin accumulation, the nematodes were pre-incubated in levamisole solution (60 µg/mL), and then analyzed by fluorescence microscope (excitation wavelength being 405 nm, and emission wavelength being 425–475 nm). For each experimental group, at least 40 nematodes were photographed, and the fluorescence intensity was measured by the Image J software.

RNA extraction and qRT-PCR

The purified total RNA was extracted by Trizol reagent, and then reverse-transcribed by using the Fastking RT Kit (Tiangen, Beijing, China). The concentrations of the extracted RNA were examined and then purified by Nanodrop (NC2000, Thermo Scientific, MA, USA). Using Fastking RT kit to synthesis cDNA, and the qRT-PCR was conducted by using LightCycler 480 with SYBR^®^ Green PCR kit (Roche, Germany) according to the manufacturer’s instructions. Primers used for cDNA amplification of the stress-related genes were shown in Table S1. The results were analyzed by using the 2^–△△Ct^ method and expressed as the relative gene expression. All gene analyses for each treatment were carried out in triplicate.

Metabolomic analysis

For the metabolomics analysis, about 10,000 nematodes were ground for 6 min (-10 °C, 50 Hz) with 400 µL of 80% methanol (v/v) that containing 0.02 mg/mL of internal standard. The samples were extracted by low-temperature sonication for 30 min (5 °C, 40 kHz) and refrigerated for another 30 min at -20 °C before centrifuging (13000 rpm, 15 min, 4 °C), then the supernatant was analyzed by UHPLC-Q Exactive HF-X (Shimadzu, Japan) that equipped with ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters, Milford, USA). Mobile phases were 5% acetonitrile aqueous solution (A) and the mixed aqueous solution (B) of acetonitrile (47.5%) and isopropyl alcohol (47.5%). The samples (2 µL) were ionized by electrospray ionization at 40 °C, and the mass spectrometry signals were collected in positive and negative ion scanning modes, respectively.

Statistical analyses

The statistical analyses were conducted by using SPSS Version 18.0 (SPSS Inc., Chicago, USA). Data were expressed as mean ± standard error of the mean (SEM) when compared to the control. Significant differences were performed by analysis of variance (ANOVA) and the LSD post hoc test. Probability levels of 0.05 and 0.01 were considered statistically significant.

MetaboAnalyst 4.0 was used for the principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) of the metabolomics data (Zhou et al. 2016). Variable importance in projection (VIP) score and ANOVA were used to select significant markers between the groups with the threshold greater than 1, and p < 0.05 were considered significant.

Results and discussion

Changes in physiological indexes of C. elegans

As a model organism, one of the biggest advantages of C. elegans is the sensitive nervous system. The changes in movement of C. elegans due to the neurological effects of the contaminants are easily to be observed in the acute behavioral tests (Anderson et al. 2004). Meanwhile, the changes in locomotion behaviors (head thrashes, body bends) can represent the neurobehavioral deficit and reflect the neurotoxicity condition of C. elegans, and serve as an indicator in assessing the effects of neurotoxic contaminants at the sublethal cellular level (Meyer and Williams 2014). In addition, body length is directly related to the development of C. elegans. Therefore, the changes in physiological indexes of C. elegans can reflect the neurotoxic effects of antibiotics indirectly.

Prolonged exposure

After the prolonged exposure to MOX at concentrations higher than 20 mg/L, the frequency of head thrashes (inhibition rate: 4.4%-8.5%) and body bend (inhibition rate: 11.6%-36.3%) of C. elegans significantly decreased (Fig. 1A and B). The trace amount of Cu^2+^ significantly inhibited head thrashing in C. elegans (23.1% inhibition), which was weakened in the presence of MOX (12.4–15.2% inhibition). When exposure to MOX-Cu (Fig. 1C), the body length of C. elegans increased slightly in the condition of lower concentration (< 20 mg/L) of MOX, while the inhibitory effects (inhibition rate: 6.4%-9.1%) were observed when the concentration of MOX was higher (≥ 20 mg/L), which was closed to that of Cu^2+^ (7.8%).

Fig. 1. Effects of MOX, Cu, and MOX-Cu on locomotion behaviors of C. elegans after prolonged exposure. A Head thrashes. B Body bends. C Body length. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups (*p < 0.05, **p < 0.01)

Chronic exposure

Nevertheless, different from the results of prolonged exposure experiments, the locomotion behaviors of C. elegans were not significantly affected when chronic exposure to MOX, Cu, and MOX-Cu (Fig. 2A and B). As the exposure time increased from 72 h to 10 days, the inhibition rate of Cu^2+^ on body length was increased from 7.8% to 13.9%, and the concentration of MOX that showed significant inhibitory effects (5.8%) on the body length decreased to 2 mg/L. Meanwhile, the inhibition rate of MOX-Cu on body length was about 11.5% when the concentration of MOX was 0.2 mg/L, and almost unchanged as the concentration of MOX continued to increase (Fig. 2C). The results indicated that the toxic effect of Cu^2+^ and MOX on body length of C. elegans was increased with the exposure time.

Fig. 2. Effects of MOX, Cu, and MOX-Cu on locomotion behaviors of C. elegans after chronic exposure. A Head thrashes. B Body bends. C Body length. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups (*p < 0.05, **p < 0.01)

In the prolonged exposure experiments, both the locomotion behaviors and growth of C. elegans were promoted as the concentration of MOX was lower than 2.0 mg/L, and inhibited as the concentration of MOX was higher than 20.0 mg/L. There was a nonlinear dose-response pattern when prolonged exposure to MOX, and lower concentrations (0.2-2 mg/L) exerted the opposite effects of higher concentrations (20–200 mg/L), exhibiting biphasic responses. Similar phenomena were observed in previous research, after 48 h exposure, the root growth of onion was promoted with 5 mg/L of MOX and inhibited as the concentration increased to 75 mg/L (Maffessoni et al. 2021).

However, the impacts of MOX on locomotion behaviors of C. elegans were decreased as the exposure time increased from 72 h to 10 days. As the basis of locomotion behavior, the neural circuits are highly fault-tolerant to a variety of perturbations, including injury and sudden changes in external environment. The invertebrates can regain the locomotion behavior when suffer with severe neurological damage, due to the neural regeneration and other physiological mechanisms (Haspel et al. 2021). The recovery of locomotion behaviors after chronic exposure to 20–200 mg/L of MOX was exactly the embodiment of fault-tolerant mechanism, which might be also indicated the delay of nervous system aging.

Similar variation trend of locomotion behaviors was observed when exposure to Cu and MOX-Cu, indicating the diminishing of neurotoxicity with the increasing of exposure time under the regulation of fault-tolerant mechanism. However, more severe inhibitory effect on growth of C. elegans was observed in chronic exposure experiment, indicating the chronic toxicity of Cu, MOX, and MOX-Cu. Meanwhile, the inhibitory effect of MOX-Cu on C. elegans growth was stable, which did not change along with the increase of MOX concentration. The phenomenon may be related to the interaction between MOX and Cu^2+^. Fluoroquinolone antibiotics can form zwitterion coordinated with Cu^2+^ through the pyridone carbonyl oxygen and carboxylate oxygen, or anionic bidentate ligand coordinated through the pyridone carbonyl oxygen and carboxylate oxygen (Nedeljković et al. 2021), which may significantly affect their biological activity.

Comparison of prolonged and chronic exposure revealed that exposure duration substantially altered the physiological toxicity in C. elegans (Figs. 1 and 2). For the prolonged exposure, locomotion behaviors responded sensitively to MOX and MOX-Cu, as reflected by significant inhibition of head thrashes (up to 8.5%) and body bends (up to 36.3%) at higher concentrations (Fig. 1A and B), with relatively moderate growth inhibition (Fig. 1C). In contrast, chronic exposure did not significantly affect locomotion (Fig. 2A and B), but led to persistent inhibition of body length at 5.8% for MOX and 11.5% for MOX-Cu, respectively (Fig. 2C). These results indicated that prolonged exposure mainly caused transient neurobehavioral disturbances, whereas chronic exposure preferentially resulted in cumulative growth inhibition.

Oxidative stress in C. elegans

ROS are potentially toxic, but they are also signaling molecules that modulate aging in diverse model organisms including C. elegans (Antonio and Elizabeth 2017). Lipofuscin is a peroxidation product of cellular components, usually used as an endogenous auto-fluorescent biomarker of oxidative stress. Antioxidant defense systems and mechanisms are necessary for organisms to regulate ROS level and protect cells that in stress condition (Baldensperger et al. 2024). Therefore, the effects of MOX and MOX-Cu on the generation of intracellular ROS and the accumulation of lipofuscin were analyzed with the aim to reveal the oxidative stress in C. elegans.

Prolonged exposure

The production of ROS significantly increased after prolonged exposure to 20 mg/L and 200 mg/L of MOX compared with that of control group, while it significantly decreased when exposed to Cu and MOX-Cu (Fig. 3A and B). A little increase in ROS level was observed when exposure to 0.2 mg/L and 2 mg/L of MOX, which may be attributed to the defenses in response to ROS-dependent damage (Desjardins et al. 2017). When exposure to 20 mg/L and 200 mg/L of MOX, the accumulation of lipofuscin (Fig. 3C and D) was approximately 1.9 times and 3.0 times higher than that of control group, respectively. Meanwhile, the accumulation of lipofuscin was about 2.1 times higher than that of control group when exposure to the combination of MOX (200 mg/L) and Cu (0.0001 mg/L).

The results suggested that MOX induced low-level stress, culminating in increased stress resistance and longevity, which was the adaptive response that resembled a process named as hormesis. Hormesis is regarded as a biphasic dose-response, the contaminant might be beneficial to livings at low dose but harmful at high doses. It can induce an adaptive defensive response during developmental stage and enhance oxidative stress resistance, and ultimately increase lifespan of C. elegans, showing a retrograde response (Kumsta et al. 2017).

ROS and Lipofuscin can not only reflect the levels of oxidative stress, but also aging indicators of C. elegans (Kim et al. 2013). The significant increase of ROS generation and lipofuscin accumulation when exposure to 20 mg/L and 200 mg/L of MOX implied the oxidative stress of C. elegans, and the physiological indexes would be inhibited consequently (Yu et al. 2021; Zhang et al. 2021). It has been reported that, a short-term oxygen free radical treatment can cause immediate and reversible changes, such as mobility reduce and growth inhibition (Back et al. 2012). Obvious antagonistic effect between Cu and MOX was obtained on the ROS generation and lipofuscin accumulation due to the generation of MOX-Cu complex, which was consistent with the results of physiological experiments. Compared with the exposure experiments of MOX or Cu, C. elegans showed less physiological fluctuation when exposure to MOX-Cu.

Fig. 3. The oxidative stress of C. elegans after prolonged exposure to MOX, Cu, and MOX-Cu. A Representative images about ROS production. B ROS production. C Representative images about lipofuscin accumulation. D Lipofuscin accumulation. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups (*p < 0.05, **p < 0.01)

Chronic exposure

The accumulation of ROS and lipofuscin were significantly reduced when chronic exposed to 20 mg/L and 200 mg/L of MOX, which might be associated with the antioxidant defenses of C. elegans (Fig. 4). Similar phenomena were observed after chronic exposed to MOX-Cu as the concentration of MOX was in the range of 0.2–20 mg/L. However, as the concentration of MOX increased to 200 mg/L, the generation of ROS and accumulation of lipofuscin significantly increased to 1.1-fold and 1.5-fold, respectively.

The cells of C. elegans will activate antioxidant defense mechanisms to relieve the damage (Van and Hekimi 2010). Oxidative stress was observed in zebrafish after 3 days exposure to 5–500 µg/L of enrofloxacin, but the activities of antioxidant enzymes almost unchanged at day 7 and 14, suggesting the amelioration of oxidative stress (Sehonova et al. 2019). Similarly, chronic exposed to MOX (20 and 200 mg/L) and MOX_(0.2−20 mg/L)-Cu(0.0001 mg/L)_ could stimulate antioxidant defense and alleviate oxidative stress in C. elegans, which might further ameliorate the locomotion behaviors of C. elegans (Chen et al. 2019).

However, it should be noting that chronic exposure to MOX and MOX-Cu had inhibited the growth of C. elegans, showing a trade-off effect, which was the strategy for balancing energy input in adapting to adverse environments. C. elegans will allocate more energy from growth to antioxidant response (Yu et al. 2018). In addition, research results indicated that a lower ROS level was associated with longer life in multicellular organisms (Zhang et al. 2021), and this conclusion was confirmed by the significant reduction of lipofuscin accumulation in C. elegans after chronic exposure to MOX (20 and 200 mg/L) and MOX_(0.2−20 mg/L)-Cu(0.0001 mg/L)_.

Fig. 4. The oxidative stress of C. elegans after chronic exposure to MOX, Cu, and MOX-Cu. A Representative images about ROS production. B ROS production. C Representative images about lipofuscin accumulation. D Lipofuscin accumulation. Data were expressed as the mean ± SEM. Asterisks indicate statistically significant differences between the exposure and control groups. (*p < 0.05, **p < 0.01)

Stress-related genes expression in C. elegans

As a common molecular level indicator, gene expression is more sensitive and specific than other indicators (Kennedy 2008). The expression of genes that associated with locomotion behaviors, growth, and antioxidant defense in C. elegans were examined (Fig. 5). Different regulation was defined when the threshold was 0.5-fold (down-regulation) or 2.0-fold (up-regulation) and the p-value was smaller than 0.05, whereas the genes were considered as slightly different in the case of without reaching the threshold (Baberschke et al. 2015). The normalized gene expression data of the tested stress-related and motor nerve-related genes after prolonged and chronic exposure to MOX and MOX-Cu were presented in Table S2-S5.

Fig. 5. Expression of stress-related genes in C. elegans after prolonged and chronic exposure to MOX (A) and MOX-Cu (B). Values of stress-related gene expression were normalized using actin mRNA and represent means (n = 3) relative to the control

Genes expression after MOX exposure experiment

1. Oxidative stress related genes

As the first line of antioxidant defenses, superoxide dismutase (SOD) could protect C. elegans from oxidative stress and prolong life-span (Van and Hekimi 2010). The gene expressions of sod-1 and sod-3 were studied due to their coding function of mitochondrial matrix MnSOD and CuZnSOD (Huang et al. 2021). As shown in Fig. 5A, the gene expression of sod-3 (0.24–1.04 fold) was decreased after prolonged exposure to MOX. The increase of ROS level and down-regulation of antioxidant genes might be attributed to the inhibition of antioxidant defenses (Shi et al. 2013). When the exposure time was extended to 10 days, the expressions of sod-1 (1.98–10.48 fold) and sod-3 (0.18–6.35 fold) were increased and ROS levels were reduced, which indicated the protective mechanism was triggered, and consequently alleviated the growth and activity of C. elegans. Similarly, the extracts of Uncaria tomentosa showed neuroprotective effect on C. elegans due to the attenuation of oxidative stress (Yu et al. 2016). Chronic treatment of Clove essential oil could induce the expressions of sod-3 or gst-4, thus extend the lifespan and promote the production and health of C. elegans (Zhang et al. 2021).

• (2)  Heat shock protein genes

Compared with the prolonged exposure experiments, the toxic effects of MOX were alleviated after chronic exposure, which might be related to the up-regulation of heat shock proteins (HSPs). HSPs were found to assist in the repair of protein damage (Morimoto et al. 1998), and the up-regulation of HSPs might protect the tissue and cell in a wide variety of stress conditions (Qian et al. 2015). Among the HSPs, hsp-16 was one of the small chaperone proteins, which could improve proteostasis and suppress proteotoxicity, avoid cell damage from stress and inhibit the apoptosis pathway, thereby conferring the longevity of C. elegans (Kumsta et al. 2017). After prolonged exposure to MOX, the expression of hsp-16 was significantly inhibited compared with the control group, including hsp-16.1 (0.01–0.37 fold), hsp-16.2 (0.07–0.47 fold) and hsp-16.48 (0.06–0.15 fold). However, the expressions of hsp-16.1 (6.85–14.95 fold), hsp-16.2 (3.79–28.12 fold), hsp-16.48 (5.14–75.58 fold) were increased after chronic exposed to MOX, which could further protect cells and tissues of C. elegans. The induction of hsp-16 could reduce the level of accumulated damage by activation repair mechanisms, ultimately slow aging and prolong lifespan of C. elegans (Jensen et al. 2006).

• (3)  Dauer formation protein 16 (daf-16)

The daf-16 was the major downstream target of the insulin/IGF-1 signaling pathway (IIS) in C. elegans, which acted as the forkhead Box O (FoxO) transcription factor to regulate lifespan and resistance (Tissenbaum 2018). The daf-16 target genes included sod-3 (being contributed to the oxidative stress response), and hsp-16 (being involved in the heat shock response) (Sun et al. 2020). The significant expressions increase of these genes suggested that the lifespan-prolonging might be mediated by daf-16 (Combes et al. 2003). After prolonged exposure to MOX, the expression of daf-16 (0.04–0.33 fold) was significantly inhibited, compared with the control group. However, the expressions of daf-16 (1.31–11.53 fold) significantly increased after chronic exposure to higher concentrations (20 and 200 mg/kg) of MOX, which might be beneficial to longevity of nematode C. elegans.

• (4)  Acetylcholinesterase genes

Acetylcholinesterase (AChE) encoded by multiple ace genes could promote neuron development and nerve regeneration in C. elegans. Among these genes, ace-1 and ace-2 account for approximately 95% of all AChE activities in C. elegans (Combes et al. 2001). The ace-1 gene is expressed in all outer epidermal cells and vaginal pump muscle cells, whereas ace-2 gene is mainly expressed in neurons of C. elegans (Baruah et al. 2014). Prolonged exposure to MOX significantly reduced the gene expressions of ace-2 (0.05–0.41 fold), ace-3 (0.24–0.60 fold), and ace-4 (0.35–0.56 fold), thereby affecting locomotion behaviors of C. elegans. However, the expressions of those genes increased with exposure time, indicated the improvements in locomotion behaviors of C. elegans.

• (5)  Apoptosis-related genes

Germline apoptosis would be activated by cep-1 in response to genotoxic stress of C. elegans, similar to its mammalian counterpart, tumor suppressor p53 (Derry et al. 2001). Previous study indicated that cep-1 could regulate multiple stress responses in the soma, and mediate apoptosis and meiotic chromosome segregation in the germline (Arum and Johnson 2007). Meanwhile, the expression of cep-1 was inversely associated with the lifespan of C. elegans (Kawasaki et al. 2010). The gene expressions of cep-1 (1.15–3.91 fold) and ape-1 (0.50–2.29 fold) were significantly increased after prolonged exposure to MOX, while the expression of cep-1 (0.37–0.55 fold) was decreased with the exposure time, which might be the reason for lifespan increase of C. elegans. The results were consistent with the changes in lipofuscin.

Overall, the expressions of HSPs, ace genes, and daf-16 were inhibited after prolonged exposure to MOX. Although the expressions of HSPs and SOD genes significantly increased under the same MOX concentration after chronic exposure, the expression of p53 protein-like gene was inhibited, compared with the prolonged exposure experiments. These results indicated that the antioxidant defenses were enhanced after chronic exposure to MOX, and the increased expression of daf-16 and its target genes (sod-3 and hsp-16), accompanied with the down-regulation of p53 protein-like gene, might be the potential molecular mechanisms for the lifespan extension in C. elegans.

Genes expression after MOX-Cu exposure experiment

The expressions of related genes in C. elegans when exposed to MOX-Cu were further investigated (Fig. 5B). The transcription of sod-1 (0.09–0.83 fold) and daf-16 (0.01–0.09 fold) decreased after prolonged exposure to MOX-Cu in all concentration combinations. Meanwhile, the down-regulation or slight repression of HSPs was observed, including hsp-16.1 (0.001–1.16 fold), hsp-16.2 (0.02–0.19 fold), and hsp-16.48 (0.13–1.07 fold). Similarly, the transcription of most acetylcholinesterase genes also showed a repression or slight repression, including ace-2 (0.01–0.07 fold), ace-3 (0.000-0.15 fold), and ace-4 (0.004–0.16 fold). However, the expression of cep-1 (8.62–18.15 fold), ape-1 (6.65–16.01 fold), and ace-1 (1.29–6.54 fold) was significantly increased compared with the control group.

The expressions of daf-16 (2.65–4.47 fold) and sod-1 (1.13–1.63 fold) was increased after chronic exposure to MOX_(0.2−20 mg/L)-Cu(0.0001 mg/L)_ due to the antioxidant defense of C. elegans, which could explain the decrease in ROS generation and lipofuscin accumulation. Meanwhile, the expression decrease of other stress-related genes (Table S6) was actually an energy-saving mode, which could provide additional resources for longevity and antioxidant defense (Baberschke et al. 2015). However, the expression of ape-1 (2.24 fold) was significantly increased as the concentration of MOX reached 200 mg/L due to oxidative stress. Although the antioxidant defense was observed when chronic exposure to MOX (20 and 200 mg/L) and MOX_(0.2−20 mg/L)- Cu(0.0001 mg/L)_, the growth of C. elegans were significantly inhibited, which might be associated with the expression increase of daf-16. Similarly, previous research results indicated that apigenin could activate daf-16 and in turn inhibited the growth of C. elegans (Hong et al. 2021).

Metabolic profiles of C. elegans

In order to investigate the mechanism of the antioxidant response in depth, metabolomics analysis of C. elegans was performed after chronic exposure to 20 mg/L of MOX and 0.0001 mg/L of Cu^2+^. Based on the metabolites detection by using LC/MS, clustering behavior between groups and the metabolites that responsible for the significance of the group were further investigated.

Fig. 6. Principal component analysis (PCA) score plot of C. elegans exposed to MOX-Cu. A Positive ion. B Negative ion

As shown in the PCA score plot (Fig. 6), a clear separation can be observed among control, MOX_(20 mg/L), and MOX(0.2−20 mg/L)-Cu(0.0001 mg/L)_ exposure groups. The metabolic properties of Cu^2+^ (0.0001 mg/L) were close to the control group due to its low concentration. In addition, biological pathways that affected by MOX-Cu was analyzed, and the relationship between the compounds could be investigated by the networks of the biological pathways. The importance of the pathway was estimated through enrichment analysis with pathway topology analysis and degree for a node. As a result, purine metabolism, arginine biosynthesis, pyrimidine metabolism, glutathione metabolism, arginine and proline metabolism, and lysine degradation were identified as the significantly affected metabolic pathways (Fig. 7).

Fig. 7. Pathway analysis of C. elegans between the control and MOX exposure group (A), Cu exposure group (B), and MOX-Cu exposure group (C)

The altered metabolites (Fig. 8) were mainly divided into 4 classes, including amino acid metabolites, nucleotide metabolisms, metabolisms of other amino acids, and membrane transports. Compared with the control group, MOX and MOX-Cu exposure resulted in the increase of amino acids such as serine, arginine, and glutamine. It has been reported that the level of specific amino acids can prolong the lifespan of C. elegans (Liu et al. 2019). Serine acts as a key substance in purine biosynthesis and one-carbon metabolic cycles, which will alleviate oxidative stress and extends lifespan via supporting methionine cycle of C. elegans (Ma et al. 2016). Glutamine and glutamate are converted to ornithine and then to arginine. Arginine exerts a significant antioxidant activity and improves resistance of C. elegans to environment stresses based on its free radical scavenging ability and regulation of IIS pathway (Edwards et al. 2015). These amino acids are all associated with the adaptive transcriptional response of DAF-16/FOXO, regulating the expressions of daf-16. The level increase of amino acids combined with the changes in the related metabolic pathways, indicating the promotion effects on antioxidant defense and lifespan of C. elegans.

In contrast to previous studies that reported physiological or biochemical responses to MOX (Huang et al. 2023), this study further clarified the overall regulatory effects of MOX and MOX-Cu in C. elegans based on the integrating metabolomic, genetic, biochemical, and phenotypic data. Compared with MOX, the co-exposure to MOX and Cu^2+^ resulted in weakened oxidative stress and altered physiological responses, which was evidenced by the reduced ROS generation and lipofuscin accumulation (Figs. 3 and 4). At the molecular level, MOX-Cu exposure induced a distinct regulation pattern of stress-responsive genes, characterized by the up-regulation of antioxidant-related genes (sod-1 and daf-16) and the suppression of apoptosis-related genes (Fig. 5). Meanwhile, the metabolomic analysis revealed that MOX-Cu mainly affected amino acid-associated metabolic pathways, including glutathione metabolism, arginine biosynthesis, and purine metabolism, which were closely associated with redox regulation and energy homeostasis (Figs. 7 and 8). These results indicated that trace Cu^2+^ modified MOX-induced toxicity through coordinated transcriptional and metabolic regulation, thereby reshaped the oxidative stress response and physiological outcomes in C. elegans.

Fig. 8. Affected metabolites of C. elegans after exposure to MOX-Cu

Conclusion

This study systematically elucidated the antioxidant responses and metabolic regulation of C. elegans under combined exposure to MOX and trace Cu^2+^ at physiological, molecular, and metabolic levels. The results showed that MOX, alone or with Cu^2+^, caused oxidative stress, and growth and locomotion impairments, which were closely associated with the dysregulation of stress-responsive, neuromuscular-related, and apoptosis-related genes. Meanwhile, the coordinated activation of antioxidant pathways and the remodeling of amino acid-associated metabolic pathways suggested the adaptive regulatory response that weakened cellular damage under MOX-Cu exposure. These results in the present work can provide scientific basis and mechanistic insights for the comprehensively environmental risk assessment of antibiotics and combined pollutants, especially for the potential impacts on the production and application of bioresources and bioprocessing systems.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1.