Kidney biomarkers in MCPA-induced acute kidney injury in rats: Reduced clearance enhances early biomarker performance

For improved early detection and assessment of severe acute kidney damage following accidental or intentional ingestion of the herbicide MCPA, we compared a panel of 14 novel kidney injury biomarkers with plasma creatinine. Male Wistar rats received four different oral doses of MCPA and plasma and urine biomarker levels were measured at 8, 24 and 48 h after MCPA exposure. Diagnostic performances using absolute levels, urine levels normalized to urine creatinine or urinary excretion rate were determined by ROC analysis. Plasma creatinine remained the best early biomarker for predicting histological changes at 48 h. The performance of plasma cystatin C in mirroring kidney function was similar to that of plasma creatinine. While urine concentrations were generally less predictive, normalization by urine creatinine greatly improved the performance of several biomarkers. This may be due to an apparent amplification of the biomarker signal on normalizing to creatinine, in the presence of a declining glomerular filtration rate prior to reaching steady state. Normalized 8 h osteopontin and albumin concentrations outperformed other normalized biomarkers in predicting histological changes at later times. Normalized urinary kidney injury molecule-1 at 48 h also correlated well with the degree of kidney damage.     

Evaluation of fibronectin 1 in one dried blood spot and in urine after rhGH treatment

Since the appearance of recombinant human growth hormone (rhGH) in the 1980s, its expansion and acquisition through the black market has increased, so the detection of its abuse continues to be a challenge. New biomarkers that are more reliable and sensitive, allowing a larger detection window, are still needed. In this line, Fibronectin 1 (FN1) has been proposed as a potential genetic and protein biomarker of rhGH abuse in peripheral blood lymphocytes, serum, and plasma. However, logistic problems associated with current blood collection in sports drug testing point towards potential new alternative matrices that could be good candidates to be evaluated. Results obtained in this study showed high ELISA FN1 levels in one dried blood spot and in urine samples in ten healthy male volunteers treated with rhGH. Results showed that especially dried blood spots appear as a potential good matrix to detect rhGH abuse by means of FN1 biomarker. Copyright © 2016 John Wiley & Sons, Ltd.     

Physiologically based toxicokinetic modeling of inhaled ethyl tertiary-butyl ether in humans.

A physiologically based toxicokinetic (PBTK) model was developed for evaluation of inhalation exposure in humans to the gasoline additive, ethyl tertiary-butyl ether (ETBE). PBTK models are useful tools to relate external exposure to internal doses and biological markers of exposure in humans. To describe the kinetics of ETBE, the following compartments were used: lungs (including arterial blood), liver, fat, rapidly perfused tissues, resting muscles, and working muscles. The same set of compartments and, in addition, a urinary excretion compartment were used for the metabolite tertiary-butyl alcohol (TBA). First order metabolism was assumed in the model, since linear kinetics has been shown experimentally in humans after inhalation exposure up to 50 ppm ETBE. Organ volumes and blood flows were calculated from individual body composition based on published equations, and tissue/blood partition coefficients were calculated from liquid/air partition coefficients and tissue composition. Estimates of individual metabolite parameters of 8 subjects were obtained by fitting the PBTK model to experimental data from humans (5, 25, 50 ppm ETBE, 2-h exposure; Nihlén et al., Toxicol. Sci., 1998; 46, 1-10). The PBTK model was then used to predict levels of the biomarkers ETBE and TBA in blood, urine, and exhaled air after various scenarios, such as prolonged exposure, fluctuating exposure, and exposure during physical activity. In addition, the interindividual variability in biomarker levels was predicted, in the eight experimentally exposed subjects after a working week. According to the model, raising the work load from rest to heavy exercise increases all biomarker levels by approximately 2-fold at the end of the work shift, and by 3-fold the next morning. A small accumulation of all biomarkers was seen during one week of simulated exposure. Further predictions suggested that the interindividual variability in biomarker levels would be higher the next morning than at the end of the work shift, and higher for TBA than for ETBE. Monte Carlo simulations were used to describe fluctuating exposure scenarios. These simulations suggest that ETBE levels in blood and exhaled air at the end of the working day are highly sensitive to exposure fluctuations, whereas ETBE levels the next morning and TBA in urine and blood are less sensitive. Considering these simulations, data from the previous toxicokinetic study and practical issues, we suggest that TBA in urine is a suitable biomarker for exposure to ETBE and gasoline vapor.     

Use of a Novel Peripheral Biomarker,Urine Trans,Trans, Muconic Acid,for Benzene Toxicity Monitoring

Benzene is a colorless poisonous toxic liquid with a sweet odor. Benzene exposure is of particular concern because recent research indicates that benzene exposure can result in chronic toxicity. Potential metabolic mechanisms underlying the hemopoietic toxicity of benzene include bioactivation of phenolic metabolites of benzene by myeloperoxidases in bone marrow, which results in hydroquinolone, a major leukemogen. In exposure and risk evaluations, the monitoring of benzene by peripheral biomarker has several advantages over a technical assessment of exposure. Of several monitoring methods, the peripheral urine biomarker, urine trans, trans‐muconic acid (ttMA) level is accepted as a novel and useful monitoring tool for early diagnosis of exposure. Presently, it is introduced as an alternative to the classical biomarker, urine phenol. Here, some previous reports on the use of ttMA as biomarker have been reviewed.     

Identification of potential biomarkers in urine and plasma after consumption of tobacco product in horses

The use of nicotine stimulants in horses is generally banned in horse racing and equestrian sports—accidental consumption of tobacco products is one of the possible causes of nicotine exposure in horses. The authors recently reported a comprehensive metabolic study of nicotine in equines, differentiating between nicotine exposure and sample contamination by means of a nicotine biomarker trans-3′-hydroxycotinine. To identify potential biomarkers for the differentiation of genuine nicotine administration and consumption of tobacco products, tobacco leaves (equivalent to 250 mg of nicotine) were nasoesophageally administered to three thoroughbred mares. Quantification methods of anatabine in plasma and urine were newly developed and validated and successfully applied to postadministration samples. Previously reported simultaneous quantification methods of eight target analytes including nicotine and its metabolites in plasma and urine were also applied to the samples. The results demonstrate that both trans-3′-hydroxycotinine and anatabine could be used as potential biomarkers in equine urine and plasma to indicate recent exposure to tobacco products in horses. As well, trans-3′-hydroxycotinine had the longest half-life as a detectable metabolite in urine and plasma. To our knowledge, this is the first report of a comprehensive study of tobacco product detection in horses.     

Military deployment human exposure assessment: urine total and isotopic uranium sampling results

Currently the Department of Defense (DoD) does not use exposure biomarkers to measure service members' exposure to environmental chemicals. Blood and urine exposure biomarkers for volatile organic compounds (VOC), selected heavy metals, depleted uranium (DU), and chemical warfare agents are currently available but have not been field tested or validated by the DoD in military deployments as a tool to document exposures. The Military Deployment Human Exposure Assessment Study, a prospective cohort of 46 soldiers deployed to Bosnia, was designed to field test blood and urine exposure biomarkers as a mechanism to document exposures to these chemicals during military deployments. Blood and urine were collected before, during, and after deployment. Standard questionnaire, environmental, and occupational monitoring data collection methods were conducted for comparison to the exposure biomarker results. This article compares and reports the pre-, during, and postdeployment urine total and isotopic uranium measurements and compares them to perceived exposures captured on questionnaire, to environmental data collected by the United Nations Environmental Program in Bosnia, and to standard U.S. urine uranium reference levels (CDC, 2003). Additionally, the questionnaire and environmental and occupational measurements are reported. The results of the study indicate that exposure biomarkers may be a valuable tool to the DoD in exposure and risk assessment with regard to environmental and occupational exposures to uranium.     

Proteomic research in renal transplantation

Expression proteomics is a valuable tool for biomarker discovery. Currently there is a great deal of interest in the development of urine biomarkers for detection of renal allograft rejection as an alternative to percutaneous needle biopsy, which is the "gold standard." Needle biopsy is costly and associated with significant patient morbidity and mortality. This review will discuss the author's current work in proteomics-based urine biomarker discovery, as well as alternative approaches used by other groups that use SELDI mass spectrometry. The current state of urine transplant biomarkers will be discussed, and in conclusion there will be a brief discussion of how urine biomarkers will be used for transplant patient management once they are validated and analyte-specific assays are developed.     

Metabonomics biomarkers for subacute toxicity screening for benzene exposure in mice

Benzene is known to produce hematotoxicity in occupational exposure workers. This study examined the utility of metabonomic biomarkers to ascertain subacute toxicity produced by benzene in male C3H/He mice. A 30-d intermittent collection of urine was obtained from mice in this experiment. The relative organ weights, blood parameters, and bone marrow smears were examined to identify specific changes of benzene-induced toxicity. In addition, an integrated analytical approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was developed to map metabolic responses in urine. Five endogenous metabolites, hypoxanthine, spermidine, 4-aminohippuric acid, indolelactic acid, and glutamylphenylalanine, were identified as potential biomarkers of benzene-induced toxicity, indicating that pathways of purine, spermidine, fatty acid, tryptophan, and peptides metabolism might be disturbed in benzene-exposed mice. Our findings showed that the use of urine metabonomics was a more sensitive tool to detect benzene-induced toxicity compared to body weight or blood parameter changes.     

Systematic review of biomonitoring studies to determine the association between exposure to organophosphorus and pyrethroid insecticides and human health outcomes

For the appropriate protection of human health it is necessary to accurately estimate the health effects of human exposure to toxic compounds. In the present review, epidemiological studies on the health effects of human exposure to organophosphorus (OP) and pyrethroid (PYR) insecticides have been critically assessed. This review is focused on studies where the exposure assessment was based on quantification of specific biomarkers in urine or plasma. The 49 studies reviewed used different epidemiological approaches and analytical methods as well as different exposure assessment methodologies. With regard to OP pesticides, the studies reviewed suggested negative effects of prenatal exposure to these pesticides on neurodevelopment and male reproduction. Neurologic effects on adults, DNA damage and adverse birth outcomes were also associated with exposure to OP pesticides. With regard to exposure to PYR pesticides, there are currently few studies investigating the adverse health outcomes due to these pesticides. The effects studied in relation to PYR exposure were mainly male reproductive effects (sperm quality, sperm DNA damage and reproductive hormone disorders). Studies' findings provided evidence to support the hypothesis that PYR exposure is adversely associated with effects on the male reproductive system. The validity of these epidemiological studies is strongly enhanced by exposure assessment based on biomarker quantification. However, for valid and reliable results and conclusions, attention should also be focused on the validity of the analytical methods used, study designs and the measured toxicants characteristics.     

Biomonitoring equivalents for deltamethrin

Measured concentrations of chemicals in blood or urine in biomonitoring studies provide an integrated reflection of exposures to chemicals via multiple routes and pathways. The potential significance of the measured concentrations of chemicals in the context of existing toxicology data and risk assessments can be assessed if chemical-specific quantitative screening criteria are available. This work presents the derivation of Biomonitoring Equivalents (BEs) for deltamethrin, a synthetic Type II pyrethroid. BEs are estimates of biomarker concentrations that are consistent with risk assessment-based exposure guidance values such as reference doses or acceptable daily intakes. BE values were derived for deltamethrin based on two biomarkers: deltamethrin in plasma and 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (DBCA), a specific metabolite, in urine. BE values for deltamethrin in plasma were based on extrapolation from measured deltamethrin concentrations in plasma in rats under conditions consistent with the Point of Departure in the critical study underlying the USEPA RfD. BE values for DBCA in urine were derived based on pharmacokinetic data from a study in human volunteers on the urinary excretion of deltamethrin and metabolites. BE values for deltamethrin in plasma corresponding to the USEPA RfD for adults and children are 20 and 2μg/L, respectively. BE values for DBCA in urine corresponding to the adult and child-specific RfDs are 50 and 7μg/L, respectively. The urinary BE value corresponding to the ADI established by the European Commission and the Joint Meeting on Pesticide Residues is 60μg/L (as DBCA in urine). These values can be used to screen biomonitoring data in the context of current risk assessments for detlamethrin.     

Disposition of lead (Pb) in saliva and blood of Sprague-Dawley rats following a single or repeated oral exposure to Pb-acetate

Biological monitoring for lead (Pb) is usually based upon a determination of blood Pb concentration; however, saliva has been suggested as a non-invasive biological matrix for assessing exposure. To further evaluate the potential utility of saliva for biomonitoring, the disposition of Pb was evaluated in whole blood (WB), red blood cells (RBC), plasma, parotid gland, bone, and saliva following either a single oral dose of 100mg Pb-acetate/kg body weight in rats or approximately 1-week after 5 sequential daily oral gavage doses of 1, 10, or 100mg Pb-acetate/kg/day. Saliva volume, pH, total saliva protein, and alpha-amylase activity were also determined. At specified times post-dosing groups of animals were anesthetized and administered pilocarpine to induce salivation. Saliva was collected, the animals were humanely sacrificed, and tissue samples were likewise collected, weighed, and processed for Pb analysis. Following a single dose exposure to Pb-acetate, Pb was detectable in all samples by 30 min post-dosing. For both the single and repeated dose treatments the concentration of Pb was highest in WB and RBC relative to plasma and saliva. However, the Pb rapidly redistributed (within 5-days post-treatment) from the blood into the bone compartment based on the substantial decrease in WB and RBC Pb concentration, and the concurrent increase in bone Pb following repeated exposure at all dose levels. Although there is clear variability in the observed Pb concentrations in plasma and saliva, there was a reasonable correlation (r(2)=0.922) between the average Pb concentrations in these biological matrices, which was consistent with previous observations. The single oral dose of Pb-acetate resulted in a decrease in salivary pH which recovered by 24h post-dosing and a decrease in alpha-amylase enzyme activity which did recover within 5-days of ceasing exposure. It is currently unclear what impact these slight functional changes may or may not have on Pb salivary clearance rates. These results demonstrate a feasibility to rapidly detect Pb in saliva and suggest that saliva may correlate best with plasma Pb concentration.     

Human ochratoxin a biomarkers--from exposure to effect

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that has received particular attention because of the toxic effects, widespread occurrence in contaminated food and feed chain, suspected causal effect on nephropathies, and, more recently, possibility of exposure by inhalation in domicile and occupational settings. Biomarkers have been used not only to ascertain the role of OTA in inducing chronic renal failure diseases, but also as a means to portray general populations' risk to the mycotoxin. Biomonitoring can thus be used to assess internal OTA exposure, with no need to recognize the main source of exposure. And so it presents undeniable advantages over the monitoring of external dose. With a just right understanding of biomarkers, it is possible to follow the trail from exposure right to effect, and so contribute both to surveillance plans and etiological studies. In recognition of the long serum half-life and the renal elimination of OTA, most of the studies present serum/plasma and/or urine analyses as markers of exposure. In this review and for each of these main matrices, a comparison over the advantages and disadvantages is offered. Although currently limited, an overview of the current knowledge on OTA biomarkers and the influential role of the individual characteristics, namely gender and age, along with season and geographical location is given. Attention is also given to the ongoing debate over the existence of OTA-DNA adducts, a biomarker of effective dose regarded as an alternative to biomarkers of internal dose. Although unspecific, OTA effect biomarkers are also reviewed.     

Human ochratoxin A biomarkers—From exposure to effect

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that has received particular attention because of the toxic effects, widespread occurrence in contaminated food and feed chain, suspected causal effect on nephropathies, and, more recently, possibility of exposure by inhalation in domicile and occupational settings. Biomarkers have been used not only to ascertain the role of OTA in inducing chronic renal failure diseases, but also as a means to portray general populations’ risk to the mycotoxin. Biomonitoring can thus be used to assess internal OTA exposure, with no need to recognize the main source of exposure. And so it presents undeniable advantages over the monitoring of external dose. With a just right understanding of biomarkers, it is possible to follow the trail from exposure right to effect, and so contribute both to surveillance plans and etiological studies. In recognition of the long serum half-life and the renal elimination of OTA, most of the studies present serum/plasma and/or urine analyses as markers of exposure. In this review and for each of these main matrices, a comparison over the advantages and disadvantages is offered. Although currently limited, an overview of the current knowledge on OTA biomarkers and the influential role of the individual characteristics, namely gender and age, along with season and geographical location is given. Attention is also given to the ongoing debate over the existence of OTA-DNA adducts, a biomarker of effective dose regarded as an alternative to biomarkers of internal dose. Although unspecific, OTA effect biomarkers are also reviewed.     

Co-exposure of cadmium and lead on bone health in a southwestern Chinese population aged 40-75 years

Both cadmium (Cd) and lead (Pb) are associated with bone health, but studies exploring the effects of Cd and Pb co-exposure on bone health are rare. This study aimed to assess the interactive effects of Cd and Pb co-exposure on bone health. In total, 799 participants, living in the targeted areas (located in southwestern China) for more than 15 years, aged 40-75 years, and subsisted on homegrown rice and vegetables were investigated. Cd and Pb levels in urine and blood samples, as well as bone mineral density, T- and Z-score were determined. After being adjusted for covariates, the T-score was negatively correlated with blood Pb in men (P < .05); for women and non-smoking women, the T-score was negatively correlated with urinary Pb (P < .05). Moreover, after being adjusted for covariates, the Z-score was negatively correlated with urinary Pb in non-smoking women (P < .05). No positive association of prevalence of osteoporosis with Cd and Pb exposure was found. However, at an additive scale, positive interactions of urinary Cd and Pb on the prevalence of osteoporosis for women and non-smoking women, and the same interactions to blood Cd and Pb for men were found. There was also a positive interaction of urinary Cd and Pb for women at a multiplicative scale. This study suggests Cd and Pb exposure could exert detrimental effects on bone health, with possible underlying interactions. Nevertheless, more studies are needed to explore the interactive effects of heavy metal co-exposure.     

Metabolic profiling of chronic cadmium exposure in the rat

A confounding problem with studying the effects of environmental exposure to contaminants in wild populations is that analytical techniques are invasive, particularly where the physiological effects of the toxin are assessed. In this study, a metabonomic approach to investigate the biochemical effects of chronic oral exposure to environmentally realistic doses of CdCl2 (low, 8 mg/kg; high, 40 mg/kg) is presented. 1H NMR spectra of urine from exposed animals were analyzed using pattern recognition methods to identify biomarkers for a 94 day exposure period. Creatinuria and both increased excretion and complexation of citrate was detected after 19 days of exposure in both exposure groups. This was accompanied by a decrease in plasma Ca2+/Mg2+ ratio in blood plasma after 94 days. Post mortem, magic angle spinning (MAS) 1H NMR spectroscopy was used alongside conventional analytical techniques to investigate intact tissue directly. According to atomic absorption spectroscopy, kidney tissue accumulated 26.8 +/- 2.5 microg of Cd2+/g dry wt (low) and 75.9 +/- 4.3 microg of Cd2+/g dry wt (high). Using high-resolution MAS 1H NMR spectroscopy altered lipid content was detected in kidneys from animals exposed to Cd2+. However, unlike acute exposure, no testicular damage was evident. This systemic approach to metabolism demonstrated the different physiological effects of chronic subacute compared with an acute exposure to Cd2+.     

Association between genetic polymorphisms in styrene-metabolizing enzymes and biomarkers in styrene-exposed workers

Single-nucleotide polymorphisms (SNP) in genes of styrene-metabolizing enzymes could modulate biomarker concentrations in blood or urine after exposure to styrene. Ten SNP were analyzed to study their influence on styrene-specific biomarkers in 89 workers of a fiber-reinforced plastic boat building factory. The internal styrene body burden was analyzed in post-shift blood and urine samples. External styrene exposure was measured by passive samplers. Spearman rank correlations between styrene exposure and biomarkers were calculated and distributions of biomarkers were checked for lognormality. Mixed linear models were applied to analyze the influence of genotypes and styrene exposure, on styrene in blood (Monday and Thursday post-shift) and on phenyglyoxylic acid (PGA; adjusted for day of measurement, Monday to Thursday) due to a lognormal distribution, smoking (current, not current), and use of respirators. Stratified analyzes for workers without and with different types of respirators were also performed. The models of both the subgroups revealed a significant influence dependent on the respirator type that workers used for inhalation protection. An influence of the external styrene concentration on the urinary PGA concentration was not observed. After implementation of the SNP into the model significant lower adjusted means of urinary PGA concentrations were found for GSTP1 105IleVal and CYP2E1 -71TT. For styrene levels in blood no significant effect was observed. A significant influence on styrene levels in blood was correlated with external styrene concentration only in workers without use of respirators. The effects of two SNP on urinary PGA decrease indicated a limited modulating SNP effect. The most effective prevention for styrene exposure was obtained with the wearing of respirators.     

Genomic and genetic biomarkers of toxicity

Biomarkers in general use today are diagnostic in that they reflect an organ's ongoing dysfunction or damage. Unfortunately, in some cases the change in the biomarker occurs too late for effective medical intervention such as seen with acute renal failure. New biomarkers of toxicity are needed to (a) alert physicians of subtle changes prior to organ dysfunction or damage to enable preventive measures and (b) predict, prior to human exposure, if a drug is likely to induce toxicity in many patients or in specific individuals. Microarray technologies can move biomarker discovery forward at an unforeseen speed as tens of thousands of genes and genetic variants can be monitored simultaneously in one biological specimen. Pharmacogenomics, herein defined as the study of a drug's effect on gene expression, can be used to discover biomarkers in solid tissues or peripheral blood cells that are altered in animals or individuals following drug exposure. Pharmacogenetics, herein defined as the study of genetic factors that affect drug response, can be employed to identify individuals whose genetic make-up suggests they would respond poorly to a particular drug. Biomarkers discovered with these approaches can result in genomic/genetic tests, protein assays or other analytical tests. Examples of such are provided with a discussion of the unresolved issues that inhibit the use of toxicity biomarkers such as biomarker validation, reimbursement of clinical tests and patient privacy.     

Physiologically based modeling of 1,2,4-trimethylbenzene inhalation toxicokinetics

A physiologically based toxicokinetic model was developed for inhalation exposure of 1,2,4-trimethylbenzene (TMB) in man. The model consists of six compartments for TMB and one compartment for the metabolite 3,4-dimethylhippuric acid (DMHA). Based on previous experimental findings from human exposures to TMB, liver metabolism was divided in two pathways, one of the first order and one of the Michaelis-Menten type. Muscle tissue was split in two compartments to account for working and resting muscle tissues during bicycle exercise. The model was used to investigate how various factors influence potential biomarkers of exposure, i.e., TMB in blood and exhaled air and DMHA in urine. Increasing the work load from rest to moderate exercise (100 W) more than doubled all biomarker levels end of shift. The effect on next morning levels was even more pronounced, illustrated by a fivefold increase in the DMHA excretion rate. Simulations of five daily 8-h exposures suggest that biomarker levels end of shift remain fairly constant whereas the levels prior to shift increase gradually during the week. This suggests that end of shift levels reflect the exposure of the same day whereas levels Friday morning reflect exposure during the entire working week. Simulations with randomly generated exposures show that the variability due to fluctuating exposure is lower next morning than end of shift. End of shift exhalation rate of TMB is more sensitive to fluctuation than TMB in venous blood and DMHA in urine. Biomarker levels for 25 ppm exposure at different sampling times are given.     

Biochemical mechanisms of nephrotoxicity: application for metabolomics

This review describes biochemical pathways of nephrotoxicity and the application of metabolic biomarkers as they relate to nephrotoxicity. Specific and sensitive biomarkers constitute the missing link in the continuum of exposure to toxins and susceptibility, disease development and possible therapeutic intervention. Important requirements for biomarker development are a detailed understanding of biochemical pathways involved in nephrotoxicity, minimal invasiveness and capacity to screen large at-risk populations. Lastly, possible biomarker candidates should be organ specific and equally applicable in preclinical drug testing as well as in clinical care of patients. This review discusses four major metabolic pathways associated with disturbed renal homeostasis: i) direct metabolic evidence of abnormal excretion of endogenous metabolites; ii) disturbances in kidney osmolarity and renal osmolyte homeostasis; iii) impaired energy state followed by dysregulation of glucose, fatty acid and ketone body metabolism; and iv) oxidative stress in renal tissues. Each of these pathways can be monitored by specific surrogate markers in urine and blood using modern metabolomics technologies.     

Sensitive biomarkers identification for differentiating Cd and Pb induced toxicity on zebrafish embryos

Cadmium (Cd) and lead (Pb) are naturally existing heavy metals that pose significant health risks. The present study aims to identify sensitive biomarkers for differentiating the toxicities induced by Cd and Pb and for providing clues for the early prediction of toxicity and environmental risk assessment. Indicators related to oxidative stress and inflammatory responses in zebrafish treated with Cd and Pb over time (from 24 hpf to 96 hpf) were compared. Furthermore, endpoints such as embryo lethality and teratogenicity were detected. Then, several related genes involved in oxidative stress and inflammatory responses characterizing both Cd and Pb exposure, along with key molecules in the MAPKs pathway, were compared at the mRNA level, allowing the selection of the most sensitive and informative biomarkers. Significant increases in reactive oxygen species (ROS) production were observed in zebrafish exposed to Cd and Pb. Cd and Pb exposure induced developmental toxicity, influencing survival rate, hatching rate, larval growth, and heart rate and causing abnormal embryonic development. Similar trends in SOD1 and SOD2 gene expression were induced by Cd and Pb, while nuclear factor erythroid-2 related factor 2 (Nrf2) gene expression responded differently to each metal. In addition, Cd and Pb induced a delayed activation of the CAT and HO-1 genes, with no apparent change in the 24 hpf and 48 hpf groups. Genes related to immunotoxicity were activated significantly in a time-dependent manner, and these genes exhibited different sensitivities to Cd and Pb. MAPKs pathway genes were also activated in a time-dependent manner, and the expression of these genes showed different effects under Cd and Pb treatment. In summary, the present works have identified some potential sensitive biomarkers. The Nrf2 gene is a potential biomarker to differentiate Pb-induced toxicity from that of Cd, and the IFN-γ gene may be used as a sensitive biomarker for evaluating the risk of Pb contamination. We found that the timeline of MAPKs pathway activation helped to differentiate these two metals toxicities. Furthermore, Pb induced the early activation of ERK2/3 and JNK1, while p38 MAPKs showed delayed activation with no apparent change in the 24 hpf group. Cd induced an early activation of ERK2 and a delayed activation of p38a, p38b, ERK3 and JNK1, indicating that the JNK1 pathway is sensitive to Pb exposure, while the p38 pathway may be susceptible to Cd. This work contributes to sensitive biomarker identification and early environmental risk evaluation for chemicals as well as toxicity prediction.