A method to determine reactive oxygen species production in intestinal and liver cell cultures using the 2',7'-dichlorodihydrofluorescein diacetate assay.

Exposure to xenobiotics can increase the production of reactive oxygen species (ROS). When detoxification organs such as the intestines and liver cannot neutralise these xenobiotics, it can induce oxidative stress and cause damage to tissues. Therefore, cell-based bioassays that indicate intracellular ROS production are a useful screening tool to evaluate the effect of these chemicals. Although flow cytometry is commonly used to measure ROS in cells, many research laboratories in the Global South do not always have access to such specialised instrumentation. Therefore, we describe a sensitive but low-cost method that can easily be used to determine ROS production in vitro. This method employs the fluorogenic dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), which emits fluorescence after being oxidised to a fluorescent derivative. Since the H2DCF-DA bioassay indicates non-specific ROS production it can be used as a marker of overall oxidative stress. This method was validated by exposing human duodenum epithelial adenocarcinoma (HuTu-80) and rat liver epithelial hepatoma (H4IIE-luc) cells to agricultural soil samples.•Production of ROS can be determined in vitro in intestinal and liver cells.•This method is inexpensive and can be easily performed in standard laboratories.•The method provides a tool for the high-throughput screening of environmental samples.

Determining superoxide dismutase content and catalase activity in mammalian cell lines.

Traditional methods for determining superoxide dismutase (SOD) content and catalase (CAT) activity rely on measuring the absorbance of individual tissue (biological) samples using a cuvette and spectrophotometer, rather than cell cultures. Although there are kits available for SOD and CAT assays, these allow for high-throughput analysis of samples and might be too expensive for research laboratories in countries from the Global South, such as South Africa. This paper describes a simple and cost-effective method to determine SOD content and CAT activity in mammalian cell cultures following exposure to environmental chemical mixtures by measuring absorbance in 96-well microplates. Moreover, the equipment used for this method is considered standard for cell culture laboratories, while the reagents and consumables are easily obtainable.•Antioxidant enzyme levels can be measured in vitro in cell cultures.•The supernatant obtained can be used to determine protein concentration, SOD content, and CAT activity.•This method is simple and affordable, allowing for the analysis of multiple samples (up to 32 samples per microplate).

The benefit of using in vitro bioassays to screen agricultural samples for oxidative stress: South Africa's case.

Applied pesticides end up in non-target environments as complex mixtures. When bioavailable, these chemicals pose a threat to living organisms and can induce oxidative stress (OS). In this article, attention is paid to OS and the physiological role of the antioxidant defense system. South African and international literature was reviewed to provide extensive evidence of pesticide-induced OS in non-target organisms, in vivo and in vitro. Although in vitro approaches are used internationally, South African studies have only used in vivo methods. Considering ethical implications, the authors support the use of in vitro bioassays to screen environmental matrices for their OS potential. Since OS responses are initiated and measurable at lower cellular concentrations compared to other toxicity endpoints, in vitro OS bioassays could be used as an early warning sign for the presence of chemical mixtures in non-target environments. Areas of concern in the country could be identified and prioritized without using animal models. The authors conclude that it will be worthwhile for South Africa to include in vitro OS bioassays as part of a battery of tests to screen environmental matrices for biological effects. This will facilitate the development and implementation of biomonitoring programs to safeguard the South African environment.

The role of effect-based methods to address water quality monitoring in South Africa: a developing country's struggle.

Water is an important resource, and it is a worldwide struggle to provide water of good quality to the whole population. Despite good governing laws and guidelines set in place to help protect the water resources and ensure it is of good quality for various consumers, the water quality in South Africa is worsening due to lack of management. The deteriorating infrastructure is becoming progressively worse, due to corruption and insufficient funds. The ever-increasing number of toxicants, as well as the identification of emerging chemicals of concern, are also challenges South Africa is facing. Chemical analysis cannot determine the total biological effect of a mixture of chemical compounds, but this shortcoming can be addressed by adding effect-based methods (EBMs) to water quality monitoring programmes. In this paper, the current status of water quality monitoring in South Africa is discussed, as well as the capacity of the country to add EBMs to its water quality monitoring programmes to protect and improve human and animal life. Created in Biorender.com.

HIV-antiretrovirals in river water from Gauteng, South Africa: Mixed messages of wastewater inflows as source.

South Africa has the highest number of people living with the human immunodeficiency virus (HIV). High usage of HIV-antiretroviral drugs (ARVs) for the treatment of the acquired immunodeficiency syndrome (AIDS) leads to the presence of ARVs in the environment. Wastewater is a major contributor of pharmaceuticals in surface and drinking water as wastewater treatment plants (WWTPs) are not designed to remove these compounds. Pharmaceuticals in the environment pose risks and the effects of ARVs on non-target organisms are largely unknown. The concentrations of ARVs in surface water upstream and downstream from WWTPs in rivers were determined. The samples were extracted by solid-phase extraction and analysed by using liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Five ARVs were quantified, mostly in downstream samples of the WWTPs, indicating wastewater as a source of ARVs, but this was not apparent in all cases. Nevirapine, lopinavir, and efavirenz were frequently detected; the highest concentrations being lopinavir and efavirenz at 38 µg/L and 24 µg/L, respectively. Aquatic ecosystems are at risk due to the constant input of pharmaceuticals that include large amounts of everyday use and the release of ARVs. This study highlights the potential of increased water pollution worldwide should more people consume increased quantities of pharmaceuticals.

Agrochemicals in freshwater systems and their potential as endocrine disrupting chemicals: A South African context.

South Africa is the largest agrochemical user in sub-Saharan Africa, with over 3000 registered pesticide products. Although they reduce crop losses, these chemicals reach non-target aquatic environments via leaching, spray drift or run-off. In this review, attention is paid to legacy and current-use pesticides reported in literature for the freshwater environment of South Africa and to the extent these are linked to endocrine disruption. Although banned, residues of many legacy organochlorine pesticides (endosulfan and dichlorodiphenyltrichloroethane (DDT)) are still detected in South African watercourses and wildlife. Several current-use pesticides (triazine herbicides, glyphosate-based herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and chlorpyrifos) have also been reported. Agrochemicals can interfere with normal hormone function of non-target organism leading to various endocrine disrupting (ED) effects: intersex, reduced spermatogenesis, asymmetric urogenital papillae, testicular lesions and infertile eggs. Although studies investigating the occurrence of agrochemicals and/or ED effects in freshwater aquatic environments in South Africa have increased, few studies determined both the levels of agricultural pesticides present and associated ED effects. The majority of studies conducted are either laboratory-based employing in vitro or in vivo bioassays to determine ED effects of agrochemicals or studies that investigate environmental concentrations of pesticides. However, a combined approach of bioassays and chemical screening will provide a more comprehensive overview of agrochemical pollution of water systems in South Africa and the risks associated with long-term chronic exposure.

May agricultural water sources containing mixtures of agrochemicals cause hormonal disturbances?

Agricultural chemicals end up in the environment as complex mixtures and it is their combinatorial effects that need to be evaluated, rather than the traditional single effect of the active ingredients. This study emphasises effects-directed analyses (androgen receptor (AR) activity) of such environmentally relevant mixtures. Soil, where glyphosate and 2,4-dichloro-phenoxyacetic acid (2,4-D) were sprayed on Bt maize, were extracted with rainwater. This allowed to test the bio-available fraction. AR effects were measured with an in vitro reporter-gene assay using MDA-kb2 cells. The cells were exposed to: single active ingredients; formulations; environmentally relevant concentrations of the active ingredients and formulations; as well as rainwater extracts. The AR was activated by rainwater extracts from soil that received a pre-and post-emergent Roundup application. The testosterone equivalents (TTEQs) derived from AR activation exceeded international drinking water trigger values. We conclude that (i) rainwater run-off from maize sprayed with Roundup and 2,4-D contained androgen active substances and (ii) the chronic exposure to this water may cause endocrine disrupting effects in humans and aquatic life which emphasise the need for intensified monitoring of environmental water resources.

Introgression of a cry1Ab transgene into open pollinated maize and its effect on Cry protein concentration and target pest survival.

In Africa, the target pests of genetically modified Bt maize are lepidopteran stem borers, notably Busseola fusca (Lepidoptera: Noctuidae). Gene flow between Bt maize hybrids and open pollinated varieties (OPVs) that do not contain the Bt trait is highly likely in areas where both types of maize are cultivated. Consequently, introgression of the cry1Ab transgene into local OPVs will result in unknown patterns of Cry1Ab protein expression in plants during follow-up seasons when recycled seed of OPVs is planted. Too low concentrations of Cry protein in such plants may result in selection for resistant alleles and accelerate resistance evolution. The aim of this study was to determine the effects of introgression of the cry1Ab transgene into an OPV, on Cry protein concentration levels and pest survival. Bt transgene introgression was done by crossing a transgenic donor hybrid containing the cry1Ab gene with a non-Bt OPV as well as with a non-Bt near-isogenic hybrid. F1 and F2 crosses as well as back crosses were done yielding 11 genotypes (treatments). Cry1Ab protein concentrations in leaf tissue of these crosses were determined by means of ELISAs. All crosses that contained the transgene had similar or higher Cry1Ab concentrations when compared to the Bt parental hybrid, except for the Bt x OPV F1-cross that had a significantly lower Cry1Ab concentration. Survival B. fusca larvae were evaluated in assays in which larvae were reared for 14 days on whorl leaf tissue of the different treatments. Larval survival did not differ between any of the maize plant treatments which contained the Bt gene. Results suggest that Bt transgene introgression into OPVs may produce plant progenies that express Cry1Ab protein at sufficient concentrations, at last up to the F2 seed, to control B. fusca larvae. Resistance evolution is however not only influenced by the frequency of pest individuals that survive exposure to the Cry proteins but also by factors such as genetics of the pest and recipient OPV, pest biology and migration behaviour.

Biological toxicity estimates show involvement of a wider range of toxic compounds in sediments from Durban, South Africa than indicated from instrumental analyses.

The toxic equivalences (TEQs) of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) from sediment of aquatic systems in Durban, South Africa were determined in two ways: 1) TEQs of PAHs and PCBs were determined by instrumental analyses and converted to 2,3,7,8­tetrachlorodibenzo­para­dioxin equivalence (TCDDeq). 2) Bioassay equivalences (BEQs) of aryl hydrocarbon receptor (AhR) ligands were analysed using the H4IIE-luc bioassay. TEQs of PCBs ranged from below limit of detection (

Fish consumption from urban impoundments: What are the health risks associated with DDTs and other organochlorine pesticides in fish to township residents of a major inland city.

Organochlorine pesticides (OCPs) in South Africa have for the most part been banned, except dichlorodiphenyltrichloroethane (DDT) which is still used as malaria vector control. The aim of this study was to determine OCP residues in the aquatic fauna of one of South Africa's most populated areas, Soweto. Risk to human health through OCP exposure via fish consumption was investigated. Clarias gariepinus was chosen as bioindicator because it is an apex predator that is in abundance, but is also a valued food source. Dichlorodiphenyltrichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), and chlordanes (CHLs) were detected in the fish tissue with the DDTs being the most prevalent at all sites. Of the three locations, Fleurhof, Orlando, and Lenasia, the latter location's fish had the highest ΣOCP load, ranging between 81 and 1190ng/gwm. The DDTs were determined to be from historic use, whereas the CHL levels indicated more recent inputs. Although the possibility of illegal use cannot be excluded completely, the presence of OCPs outside of their allowed areas of use indicate that these compounds not only stay in the aquatic systems long term, but may be of concern in areas previously not considered high risk areas. The OCP residues in C. gariepinus from the study area pose an extremely high risk to human health when consumed, and has a cancer risk as high as 1 in 10. This potential problem should be kept in consideration when developing national health and conservation strategies.

Polycyclic aromatic hydrocarbons (PAHs) in sediments from a typical urban impacted river: application of a comprehensive risk assessment.

Soweto and Lenasia, the most densely populated area of South Africa, is simultaneously a thriving metropolis, with a fair share of people still living in squalor conditions directly dependant on the natural resources. Because of industrialisation the populace and environment in this urban area are exposed to various pollutants. The aquatic environment was selected as a proxy to study the effect of industrial pollution in this area. The concentrations, source identification, and various environmental risks of polycyclic aromatic hydrocarbons (PAHs) were determined in sediments of the upper reaches of the Klip River. Composite sediment samples collected in low-flow conditions in 2013 and 2014 ranged from 270-5400 ng/g. The PAHs in this aquatic ecosystem were dominated by 4-ring congeners and could be attributed to combustion of organic fuels by chemical mass balance. Heavy traffic and industrial complexes in the northern part of the study area were responsible for the PAH fingerprints. Probable adverse effects such as toxicity to benthic biota were proven after comparison with international sediment quality guidelines (SQG) both survey years. Toxic equivalence quotients (TEQs) calculated for the sediments using fish potency factors (FPFs) were up to 30 times greater than the Canadian guideline for dioxin-like compounds, indicating high probability of carcinogenic effect to fish mediated through the aryl-hydrocarbon receptor. Finally, sediments in the area posed moderate to high ecological risk, which corroborates the other toxicity assessments. The advantage of investigating multiple risk endpoints, is the comprehensive results obtained that allows for a more realistic representation of the study area. Consequently more aspects are kept into account that results in better conclusions.

Pathogenic features of heterotrophic plate count bacteria from drinking-water boreholes.

Evidence suggests that heterotrophic plate count (HPC) bacteria may be hazardous to humans with weakened health. We investigated the pathogenic potential of HPC bacteria from untreated borehole water, consumed by humans, for: their haemolytic properties, the production of extracellular enzymes such as DNase, proteinase, lipase, lecithinase, hyaluronidase and chondroitinase, the effect simulated gastric fluid has on their survival, as well as the bacteria's antibiotic-susceptible profile. HuTu-80 cells acted as model for the human intestine and were exposed to the HPC isolates to determine their effects on the viability of the cells. Several HPC isolates were α- or ß-haemolytic, produced two or more extracellular enzymes, survived the SGF treatment, and showed resistance against selected antibiotics. The isolates were also harmful to the human intestinal cells to varying degrees. A novel pathogen score was calculated for each isolate. Bacillus cereus had the highest pathogen index: the pathogenicity of the other bacteria declined as follows: Aeromonas taiwanensis > Aeromonas hydrophila > Bacillus thuringiensis > Alcaligenes faecalis > Pseudomonas sp. > Bacillus pumilus > Brevibacillus sp. > Bacillus subtilis > Bacillus sp. These results demonstrated that the prevailing standards for HPCs in drinking water may expose humans with compromised immune systems to undue risk.

Dioxin, furan and PCB serum levels in a South African Tswana population: comparing the polluting effects of using different cooking and heating fuels.

In South Africa, 26-50% of households use solid fuel for cooking food and heating houses. When used as fuel, wood and chlorinated waste are known sources of polychlorinated dibenzo-para-dioxins, polychlorinated dibenzofurans (PCDD/Fs), and polychlorinated biphenyls (PCBs). Here, we compare PCDD/F, dioxin-like PCB (DL-PCB), and non-DL-PCB (NDL-PCB) levels in serum of 693 Tswana individuals in the North West province, who either burn solid biofuels or have access to electricity, gas, and paraffin. This is the first South African study on dioxin levels in humans with more than 100 participants. Serum was pooled according to fuel use, as well as to confounding factors such as gender and age. Solid-phase extraction was used to remove the target analytes from serum, after which the extracts were further refined automatically using a combination of multilayer sorbents. Compound concentrations were determined by high-resolution mass spectrometry after high-resolution gas chromatography. Mean serum lipid content was determined enzymatically to be 5.91 ± 0.42 g/L. The PCDD/F and DL-PCB levels were similar to global concentrations reported for non-exposed adults. The mean of the total Toxic Equivalencies (ΣTEQ) was 6.9 ± 3.3 pg/g lipid and the mean NDL-PCB was 70.1 ± 42.8 ng/g lipid. The mean concentrations of the PCDDs, PCDFs and the corresponding World Health Organization-TEQ (WHO-TEQ) of the population using electricity, gas, and paraffin were greater than of those reliant on solid biomass (p = 0), whereas the DL-PCBs, their corresponding WHO-TEQ, and NDL-PCBs were greater for the population who use biofuels but not significantly so. The females had higher serum levels of the PCDDs (p = 0) and PCDFs (not significant) whereas the PCBs were higher for the males (p = 0). Breastfeeding women presented lower levels of all compound classes than their non-breastfeeding counterparts (p=0) and older subjects manifested greater pollutant loads than the younger generation (p = 0). For our study population, being regularly exposed to the combustion of solid biofuels caused higher serum levels of DL-PCBs and NDL-PCBs but not of PCDDs and PCDFs.

Distribution profiles of selected organic pollutants in soils and sediments of industrial, residential and agricultural areas of South Africa.

Currently very little data exists on the presence of persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs) in the South African environment. To address this data gap a preliminary study of a highly industrialised area of South Africa, the Vaal Triangle, was done. Soil and sediment samples from the Vaal Triangle, as well as other areas in central South Africa, were analysed with high resolution gas chromatography-high resolution mass spectrometry (HRGC/HRMS) for the presence of PAHs, polychlorinated biphenyls (PCBs), dioxin-like chemicals and organochlorine pesticides (OCPs). Results showed that these chemicals are present in the South African environment with concentrations ranging between 39,000 ng g(-1) for SigmaPAHs and 0.01 ng g(-1) for dicofol. Principal component analysis (PCA) indicated different pollution sources in industrial and agricultural areas.

Dioxin-like chemicals in soil and sediment from residential and industrial areas in central South Africa.

Persistent organic pollutants (POPs) are a global concern due to their ubiquitous presence and toxicity. Currently, there is a lack of information regarding POPs from South Africa. Here we report and interpret concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), -dibenzofurans (PCDFs) and co-planar-biphenyls (PCBs) in soils and sediments collected from central South Africa. High resolution gas chromatography-high resolution mass spectrometry (HRGC/HRMS) and the H4IIE-luc bio-assay were used to identify and quantify individual PCDD/F congeners and to report the total concentration of 2,3,7,8-tetrachloro dibenzo-p-dioxin equivalents (TCDD-EQ), respectively. TCDD-EQs determined by use of the bio-assay, and concentrations of WHO(2005)-TEQ (toxic equivalents) determined by chemical analysis, were similar. The limit of detection (LOD) for the bio-assay was 0.82 and 2.8 ng TCDD-EQ kg(-1), dw for sediment and soil, respectively. EQ20 concentrations determined by use of the bio-assay ranged from