Jonathan K. Challis


DOI bib
A wastewater-based risk index for SARS-CoV-2 infections among three cities on the Canadian Prairie
Mohsen Asadi, Femi Francis Oloye, Yuwei Xie, Jenna Cantin, Jonathan K. Challis, Kerry N. McPhedran, Warsame Yusuf, David Champredon, Ximing Pu, Chantel De Lange, Seba El-Baroudy, Mark R. Servos, Paul D. Jones, John P. Giesy, Markus Brinkmann
Science of The Total Environment, Volume 876

Wastewater surveillance (WWS) is useful to better understand the spreading of coronavirus disease 2019 (COVID-19) in communities, which can help design and implement suitable mitigation measures. The main objective of this study was to develop the Wastewater Viral Load Risk Index (WWVLRI) for three Saskatchewan cities to offer a simple metric to interpret WWS. The index was developed by considering relationships between reproduction number, clinical data, daily per capita concentrations of virus particles in wastewater, and weekly viral load change rate. Trends of daily per capita concentrations of SARS-CoV-2 in wastewater for Saskatoon, Prince Albert, and North Battleford were similar during the pandemic, suggesting that per capita viral load can be useful to quantitatively compare wastewater signals among cities and develop an effective and comprehensible WWVLRI. The effective reproduction number (Rt) and the daily per capita efficiency adjusted viral load thresholds of 85 × 106 and 200 × 106 N2 gene counts (gc)/population day (pd) were determined. These values with rates of change were used to categorize the potential for COVID-19 outbreaks and subsequent declines. The weekly average was considered 'low risk' when the per capita viral load was 85 × 106 N2 gc/pd. A 'medium risk' occurs when the per capita copies were between 85 × 106 and 200 × 106 N2 gc/pd. with a rate of change <100 %. The start of an outbreak is indicated by a 'medium-high' risk classification when the week-over-week rate of change was >100 %, and the absolute magnitude of concentrations of viral particles was >85 × 106 N2 gc/pd. Lastly, a 'high risk' occurs when the viral load exceeds 200 × 106 N2 gc/pd. This methodology provides a valuable resource for decision-makers and health authorities, specifically given the limitation of COVID-19 surveillance based on clinical data.

DOI bib
Wastewater discharges alter microbial community composition in surface waters of the canadian prairies
Milena Esser, Cameron Hoggarth, Helen M. Baulch, Jonathan K. Challis, Yuwei Xie, John P. Giesy, Markus Hecker, Markus Brinkmann
Chemosphere, Volume 334

Microbial communities are an important component of freshwater biodiversity that is threatened by anthropogenic impacts. Wastewater discharges pose a particular concern by being major sources of anthropogenic contaminants and microorganisms that may influence the composition of natural microbial communities. Nevertheless, the effects of wastewater treatment plant (WWTP) effluents on microbial communities remain largely unexplored. In this study, the effects of wastewater discharges on microbial communities from five different WWTPs in Southern Saskatchewan were investigated using rRNA gene metabarcoding. In parallel, nutrient levels and the presence of environmentally relevant organic pollutants were analyzed. Higher nutrient loads and pollutant concentrations resulted in significant changes in microbial community composition. The greatest changes were observed in Wascana Creek (Regina), which was found to be heavily polluted by wastewater discharges. Several taxa occurred in greater relative abundance in the wastewater-influenced stream segments, indicating anthropogenic pollution and eutrophication, especially taxa belonging to Proteobacteria, Bacteroidota, and Chlorophyta. Strong decreases were measured within the taxa Ciliphora, Diatomea, Dinoflagellata, Nematozoa, Ochrophyta, Protalveolata, and Rotifera. Across all sample types, a significant decline in sulfur bacteria was measured, implying changes in functional biodiversity. In addition, downstream of the Regina WWTP, an increase in cyanotoxins was detected which was correlated with a significant change in cyanobacterial community composition. Overall, these data suggest a causal relationship between anthropogenic pollution and changes in microbial communities, possibly reflecting an impairment of ecosystem health.

DOI bib
Understanding common population markers for SARS-CoV-2 RNA normalization in wastewater – A review
Femi Francis Oloye, Yuwei Xie, Jonathan K. Challis, Oluwabunmi P. Femi-Oloye, Markus Brinkmann, Kerry N. McPhedran, Paul D. Jones, Mark R. Servos, John P. Giesy
Chemosphere, Volume 333

Wastewater monitoring and epidemiology have seen renewed interest during the recent COVID-19 pandemic. As a result, there is an increasing need to normalize wastewater-derived viral loads in local populations. Chemical tracers, both exogenous and endogenous compounds, have proven to be more stable and reliable for normalization than biological indicators. However, differing instrumentation and extraction methods can make it difficult to compare results. This review examines current extraction and quantification methods for ten common population indicators: creatinine, coprostanol, nicotine, cotinine, sucralose, acesulfame, androstenedione 5-hydroindoleacetic acid (5-HIAA), caffeine, and 1,7-dimethyluric acid. Some wastewater parameters such as ammonia, total nitrogen, total phosphorus, and daily flowrate were also evaluated. The analytical methods included direct injection, dilute and shoot, liquid/liquid, and solid phase extraction (SPE). Creatine, acesulfame, nicotine, 5-HIAA and androstenedione have been analysed by direct injection into LC-MS; however, most authors prefer to include SPE steps to avoid matrix effects. Both LC-MS and GC-MS have been successfully used to quantify coprostanol in wastewater, and the other selected indicators have been quantified successfully with LC-MS. Acidification to stabilize the sample before freezing to maintain the integrity of samples has been reported to be beneficial. However, there are arguments both for and against working at acidic pHs. Wastewater parameters mentioned earlier are quick and easy to quantify, but the data does not always represent the human population effectively. A preference for population indicators originating solely from humans is apparent. This review summarises methods employed for chemical indicators in wastewater, provides a basis for choosing an appropriate extraction and analysis method, and highlights the utility of accurate chemical tracer data for wastewater-based epidemiology.


DOI bib
Effects of aqueous fluoxetine exposure on gut microbiome of adult Pimephales promelas
Alana Weber, Yuwei Xie, Jonathan K. Challis, Abigail DeBofsky, Phillip J. Ankley, Markus Hecker, Paul D. Jones, John P. Giesy
Science of The Total Environment, Volume 813

The microbiome of the gut is vital for homeostasis of hosts with its ability to detoxify and activate toxicants, as well as signal to the immune and nervous systems. However, in the field of environmental toxicology, the gut microbiome has only recently been identified as a measurable indicator for exposure to environmental pollutants. Antidepressants found in effluents of wastewater treatment plants and surface waters have been shown to exhibit antibacterial-like properties in vitro, where some bacteria are known to express homologous proteins that bind antidepressants in vertebrates. Therefore, it has been hypothesized that exposure to antidepressant drugs might affect gut microbiota of aquatic organisms. In this study, the common antidepressant, fluoxetine, was investigated to determine whether it can modulate the gut microbiome of adult fathead minnows. A 28-day, sub-chronic, static renewal exposure was performed with nominal fluoxetine concentrations of 0.01, 10 or 100 μg/L. Using 16S rRNA amplicon sequencing, shifts among the gut-associated microbiota were observed in individuals exposed to the greatest concentration, with greater effects observed in females. These changes were associated with a decrease in relative proportions of commensal bacteria, which can be important for health of fish including bacteria essential for fatty acid oxidation, and an increase in relative proportions of pathogenic bacteria associated with inflammation. Results demonstrate, for the first time, how antidepressants found in some aquatic environments can influence gut microbiota of fishes.

DOI bib
RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern
Yuwei Xie, Jonathan K. Challis, Femi Francis Oloye, Mohsen Asadi, Jenna Cantin, Markus Brinkmann, Kerry N. McPhedran, Natacha S. Hogan, Mike Sadowski, Paul D. Jones, Chrystal Landgraff, Chand S. Mangat, Mark R. Servos, John P. Giesy
ACS ES&T Water, Volume 2, Issue 11

There are no standardized protocols for quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater to date, especially for population normalization. Here, a pipeline was developed, applied, and assessed to quantify SARS-CoV-2 and key variants of concern (VOCs) RNA in wastewater at Saskatoon, Canada. Normalization approaches using recovery ratio and extraction efficiency, wastewater parameters, or population indicators were assessed by comparing to daily numbers of new cases. Viral load was positively correlated with daily new cases reported in the sewershed. Wastewater surveillance (WS) had a lead time of approximately 7 days, which indicated surges in the number of new cases. WS revealed the variant α and δ driving the third and fourth wave, respectively. The adjustment with the recovery ratio and extraction efficiency improved the correlation between viral load and daily new cases. Normalization of viral concentration to concentrations of the artificial sweetener acesulfame K improved the trend of viral load during the Christmas and New Year holidays when populations were dynamic and variable. Acesulfame K performed better than pepper mild mottle virus, creatinine, and ammonia for population normalization. Hence, quality controls to characterize recovery ratios and extraction efficiencies and population normalization with acesulfame are promising for precise WS programs supporting decision-making in public health.

DOI bib
16S rRNA metabarcoding unearths responses of rare gut microbiome of fathead minnows exposed to benzo[a]pyrene
Abigail DeBofsky, Yuwei Xie, Jonathan K. Challis, Phillip J. Ankley, Markus Brinkmann, Paul D. Jones, John P. Giesy
Science of The Total Environment, Volume 807

Activities of gut microbiomes are often overlooked in assessments of ecotoxicological effects of environmental contaminants. Effects of the polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) on active gut microbiomes of juvenile fathead minnows (Pimephales promelas) were investigated. Fish were exposed for two weeks, to concentrations of 0, 1, 10, 100, or 1000 μg BaP g-1 in the diet. The active gut microbiome was characterized using 16S rRNA metabarcoding to determine its response to dietary exposure of BaP. BaP reduced alpha-diversity at the greatest exposure concentrations. Additionally, exposure to BaP altered community composition of active microbiome and resulted in differential proportion of taxa associated with hydrocarbon degradation and fish health. Neighborhood selection networks of active microbiomes were not reduced with greater concentrations of BaP, which suggests ecological resistance and/or resilience of gut microbiota. The active gut microbiome had a similar overall biodiversity as that of the genomic gut microbiota, but had a distinct composition from that of the 16S rDNA profile. Responses of alpha- and beta-diversities of the active microbiome to BaP exposure were consistent with that of genomic microbiomes. Normalized activity of microbiome via the ratio of rRNA to rDNA abundance revealed rare taxa that became active or dormant due to exposure to BaP. These differences highlight the need to assess both 16S rDNA and rRNA metabarcoding to fully derive bacterial compositional changes resulting from exposure to contaminants.

DOI bib
Impacts of wastewater effluents and seasonal trends on levels of antipsychotic pharmaceuticals in water and sediments from two cold-region rivers
Ana Sharelys Cardenas Perez, Jonathan K. Challis, Xiaowen Ji, John P. Giesy, Markus Brinkmann
Science of The Total Environment, Volume 851

Most pharmaceuticals are found at trace concentrations in aquatic systems, but their continuous release and potential accumulation can lead to adverse health effects in exposed organisms. Concentrations can vary temporally, driven by variations in discharges of receiving waters, sorption to sediments, and other biotic and abiotic exchange processes. The principal aim of this research was to better understand the occurrence, trends, and dynamics of pharmaceuticals in a cold-climate, riverine environment. To this end, a suite of seven representative antipsychotic pharmaceuticals was measured upstream and downstream of two wastewater treatment plants (WWTPs) in Saskatchewan, Canada, located in the South Saskatchewan River and Wascana Creek, respectively, across three seasons. Concentrations of analytes were in the ng/L range and generally greater downstream of both WWTPs compared to upstream. Some compounds, including the tricyclic antidepressant amitriptyline, which was the most abundant analyte in water and sediment from both sites and across seasons, reached low μg/L concentrations. Data collected from this research effort indicate contamination with antipsychotic pharmaceuticals, with the potential to adversely impact exposed organisms.

DOI bib
Rapid transition between SARS-CoV-2 variants of concern Delta and Omicron detected by monitoring municipal wastewater from three Canadian cities
Femi Francis Oloye, Yuwei Xie, Mohsen Asadi, Jenna Cantin, Jonathan K. Challis, Markus Brinkmann, Kerry N. McPhedran, Kevin Kristian, Mark P. Keller, Mike Sadowski, Paul D. Jones, Chrystal Landgraff, Chand S. Mangat, Meghan Fuzzen, Mark R. Servos, John P. Giesy
Science of The Total Environment, Volume 841

Monitoring the communal incidence of COVID-19 is important for both government and residents of an area to make informed decisions. However, continuous reliance on one means of monitoring might not be accurate because of biases introduced by government policies or behaviours of residents. Wastewater surveillance was employed to monitor concentrations of SARS-CoV-2 RNA in raw influent wastewater from wastewater treatment plants serving three Canadian Prairie cities with different population sizes. Data obtained from wastewater are not directly influenced by government regulations or behaviours of individuals. The means of three weekly samples collected using 24 h composite auto-samplers were determined. Viral loads were determined by RT-qPCR, and whole-genome sequencing was used to charaterize variants of concern (VOC). The dominant VOCs in the three cities were the same but with different proportions of sub-lineages. Sub-lineages of Delta were AY.12, AY.25, AY.27 and AY.93 in 2021, while the major sub-lineage of Omicron was BA.1 in January 2022, and BA.2 subsequently became a trace-level sub-variant then the predominant VOC. When each VOC was first detected varied among cities; However, Saskatoon, with the largest population, was always the first to present new VOCs. Viral loads varied among cities, but there was no direct correlation with population size, possibly because of differences in flow regimes. Population is one of the factors that affects trends in onset and development of local outbreaks during the pandemic. This might be due to demography or the fact that larger populations had greater potential for inter- and intra-country migration. Hence, wastewater surveillance data from larger cities can typically be used to indicate what to expect in smaller communities.

DOI bib
Predicting Hepatic Clearance of Psychotropic Drugs in Isolated Perfused Fish Livers Using a Combination of Two <i>In Vitro</i> Assays
Zoey M. Bourgeois, Jordan J Comfort, Matthew Schultz, Jonathan K. Challis, Jenna Cantin, Xiaowen Ji, John P. Giesy, Markus Brinkmann
Environmental Science & Technology, Volume 56, Issue 22

In vitro biotransformation assays with primary trout hepatocytes (RT-HEP) or liver subcellular fractions (RT-S9) have been proposed as valuable tools to help scientists and regulators better understand the toxicokinetics of chemicals. While both assays have been applied successfully to a diversity of neutral organic chemicals, only the RT-S9 assay has been applied to a large number of ionizable organic chemicals. Here, a combination of an in vitro biotransformation assay with RT-HEP with an active transport assay based on the permanent rainbow trout liver cell line RTL-W1 was used to qualitatively predict the potential hepatic clearance of nine psychotropic drugs with various degrees of ionization. Predictions were compared with rates of clearance measured in isolated perfused rainbow trout livers, and the importance of active transport was verified in the presence of the active transport inhibitor cyclosporin A. For the first time, it was demonstrated that a combination of biotransformation and active transport assays is powerful for the prediction of rates of hepatic clearance of ionizable chemicals. Ultimately, it is expected that this approach will allow for use of fewer animals while at the same time improving our confidence in the use of data from in vitro assays in chemical risk assessment.

DOI bib
Exposure to the Tire Rubber-Derived Contaminant 6PPD-Quinone Causes Mitochondrial Dysfunction <i>In Vitro</i>
Hannah Mahoney, Francisco C. da Silva, Catherine Roberts, Matthew Schultz, Xiaowen Ji, Alper James Alcaraz, David W. Montgomery, Summer Selinger, Jonathan K. Challis, John P. Giesy, Lynn P. Weber, David M. Janz, Steve Wiseman, Markus Hecker, Markus Brinkmann
Environmental Science & Technology Letters, Volume 9, Issue 9


DOI bib
Life Cycle Exposure to Environmentally Relevant Concentrations of Diphenyl Phosphate (DPhP) Inhibits Growth and Energy Metabolism of Zebrafish in a Sex-Specific Manner
Qiliang Chen, Xiaolong Lian, Jie An, Ningbo Geng, Haijun Zhang, Jonathan K. Challis, Yun Luo, Yaxin Liu, Guanyong Su, Yuwei Xie, Yingwen Li, Zhihao Liu, Yanjun Shen, John P. Giesy, Yufeng Gong
Environmental Science & Technology

Due to commercial uses and environmental degradation of aryl phosphate esters, diphenyl phosphate (DPhP) is frequently detected in environmental matrices and is thus of growing concern worldwide. However, information on potential adverse effects of chronic exposure to DPhP at environmentally realistic concentrations was lacking. Here, we investigated the effects of life cycle exposure to DPhP on zebrafish at environmentally relevant concentrations of 0.8, 3.9, or 35.6 μg/L and employed a dual-omics approach (metabolomics and transcriptomics) to characterize potential modes of action. Exposure to DPhP at 35.6 μg/L for 120 days resulted in significant reductions in body mass and length of male zebrafish, but did not cause those same effects to females. Predominant toxicological mechanisms, including inhibition of oxidative phosphorylation, down-regulation of fatty acid oxidation, and up-regulation of phosphatidylcholine degradation, were revealed by integrated dual-omics analysis and successfully linked to adverse outcomes. Activity of succinate dehydrogenase and protein content of carnitine O-palmitoyltransferase 1 were significantly decreased in livers of male fish exposed to DPhP, which further confirmed the proposed toxicological mechanisms. This study is the first to demonstrate that chronic, low-level exposure to DPhP can retard growth via inhibiting energy output in male zebrafish.

DOI bib
Responses of juvenile fathead minnow (Pimephales promelas) gut microbiome to a chronic dietary exposure of benzo[a]pyrene
Abigail DeBofsky, Yuwei Xie, Jonathan K. Challis, Niteesh Jain, Markus Brinkmann, Paul D. Jones, John P. Giesy
Environmental Pollution, Volume 278

The microbiome has been described as an additional host “organ” with well-established beneficial roles. However, the effects of exposures to chemicals on both structure and function of the gut microbiome of fishes are understudied. To determine effects of benzo[ a ]pyrene (BaP), a model persistent organic pollutant, on structural shifts of gut microbiome in juvenile fathead minnows ( Pimephales promelas ), fish were exposed ad libitum in the diet to concentrations of 1, 10, 100, or 1000 μg BaP g −1 food, in addition to a vehicle control, for two weeks. To determine the link between exposure to BaP and changes in the microbial community, concentrations of metabolites of BaP were measured in fish bile and 16S rRNA amplicon sequencing was used to evaluate the microbiome. Exposure to BaP only reduced alpha-diversity at the greatest exposure concentrations. However, it did alter community composition assessed as differential abundance of taxa and reduced network complexity of the microbial community in all exposure groups. Results presented here illustrate that environmentally-relevant concentrations of BaP can alter the diversity of the gut microbiome and community network connectivity. Highlights • Dominant phyla of gut microbiome are consistent with those of other freshwater fishes. • BaP metabolites and exposure doses were consistent with those found in contaminated sites. • Dietary BaP exposure has significant, dose-dependent effects on the fish gut microbiome. • Dietary BaP exposure altered association networks of gut microbiome. Environmentally-relevant concentrations of BaP can alter the diversity of the gut microbiome and community network connectivity via dietary exposure route.

DOI bib
Remodeling of Arctic char ( <i>Salvelinus alpinus</i> ) lipidome under a stimulated scenario of Arctic warming
Chao Wang, Yufeng Gong, Fuchang Deng, Enmin Ding, Jie Tang, Garry Codling, Jonathan K. Challis, Derek Green, Jing Wang, Qiliang Chen, Yuwei Xie, Shu Su, Zilin Yang, Jason C. Raine, Paul D. Jones, Song Tang, John P. Giesy
Global Change Biology, Volume 27, Issue 14

Arctic warming associated with global climate change poses a significant threat to populations of wildlife in the Arctic. Since lipids play a vital role in adaptation of organisms to variations in temperature, high-resolution mass-spectrometry-based lipidomics can provide insights into adaptive responses of organisms to a warmer environment in the Arctic and help to illustrate potential novel roles of lipids in the process of thermal adaption. In this study, we studied an ecologically and economically important species-Arctic char (Salvelinus alpinus)-with a detailed multi-tissue analysis of the lipidome in response to chronic shifts in temperature using a validated lipidomics workflow. In addition, dynamic alterations in the hepatic lipidome during the time course of shifts in temperature were also characterized. Our results showed that early life stages of Arctic char were more susceptible to variations in temperature. One-year-old Arctic char responded to chronic increases in temperature with coordinated regulation of lipids, including headgroup-specific remodeling of acyl chains in glycerophospholipids (GP) and extensive alterations in composition of lipids in membranes, such as less lyso-GPs, and more ether-GPs and sphingomyelin. Glycerolipids (e.g., triacylglycerol, TG) also participated in adaptive responses of the lipidome of Arctic char. Eight-week-old Arctic char exhibited rapid adaptive alterations of the hepatic lipidome to stepwise decreases in temperature while showing blunted responses to gradual increases in temperature, implying an inability to adapt rapidly to warmer environments. Three common phosphatidylethanolamines (PEs) (PE 36:6|PE 16:1_20:5, PE 38:7|PE 16:1_22:6, and PE 40:7|PE 18:1_22:6) were finally identified as candidate lipid biomarkers for temperature shifts via machine learning approach. Overall, this work provides additional information to a better understanding of underlying regulatory mechanisms of the lipidome of Arctic organisms in the face of near-future warming.

DOI bib
Ractopamine and Other Growth-Promoting Compounds in Beef Cattle Operations: Fate and Transport in Feedlot Pens and Adjacent Environments
Jonathan K. Challis, Srinivas Sura, Jenna Cantin, Ashley Curtis, K. M. Shade, Tim A. McAllister, Paul D. Jones, John P. Giesy, Francis J. Larney
Environmental Science & Technology, Volume 55, Issue 3

The current study represents a comprehensive investigation of the occurrence and fates of trenbolone acetate (TBA) and metabolites 17α-trenbolone (17α-TBOH), 17β-TBOH, and trendione (TBO); melengesterol acetate (MGA); and the less commonly studied β-andrenergic agonist ractopamine (RAC) in two 8 month cattle feeding trials and simulated rainfall runoff experiments. Cattle were administered TBA, MGA, or RAC, and their residues were measured in fresh feces, pen floor material, and simulated rainfall runoff from pen floor surfaces and manure-amended pasture. Concentrations of RAC ranged from 3600 ng g–1, dry weight (dw), in pen floor to 58 000 ng g–1 in fresh feces and were, on average, observed at 3–4 orders of magnitude greater than those of TBA and MGA. RAC persisted in pen floors (manure t1/2 = 18–49 days), and contamination of adjacent sites was observed, likely via transport of windblown particulates. Concentrations in runoff water from pen floors extrapolated to larger-scale commercial feedlots revealed that a single rainfall event could result in mobilization of gram quantities of RAC. This is the first report of RAC occurrence and fate in cattle feedlot environments, and will help understand the risks posed by this chemical and inform appropriate manure-management practices.


DOI bib
Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf
Mourad A. M. Aboul‐Soud, Abdelkader E. Ashour, Jonathan K. Challis, Atallah F. Ahmed, Ashok Kumar, Amr Nassrallah, Tariq A Alahmari, Quaiser Saquib, Maqsood A. Siddiqui, Yazeed A. Al‐Sheikh, Hany A. El‐Shemy, Ahmed M. Aboul‐Enein, Khalid M. AlGhamdi, Paul D. Jones, John P. Giesy
Plants, Volume 9, Issue 10

Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.