Toolbox for the sampling and monitoring of benthic cyanobacteria.

Benthic cyanobacteria are a nuisance because they produce highly potent toxins and taste and odour compounds. Despite this, benthic cyanobacteria remain far less studied than their planktonic counterparts. For example, little is known about their growth or the seasonality of their secondary metabolite production. Moreover, sampling and monitoring techniques commonly used for the survey of planktonic species are not necessarily applicable to benthic forms. This study aimed to develop and validate a new sampling device for the routine monitoring of benthic mats. Molecular monitoring techniques were established and validated on environmental samples collected in a South Australian reservoir (SA-L2). A total of eight qPCR assays were applied to samples in order to track seasonal variations in cyanobacteria concentrations and associated secondary metabolite production. Next Generation Sequencing was utilised to conduct a microbial community composition analysis and to select the most appropriate substrate material for the sampling of benthic cyanobacteria. The concentration of the secondary metabolites geosmin and 2-methyl-isoborneol were quantified using High-Performance Liquid Chromatography, and concentrations of key nutrients (N, P) were quantified in water samples. The sampling device designed proved efficient and easy to use in the field. The qPCR assay designed for the amplification of the cyanobacterial MIB synthase had a high efficiency with a minimum limit of quantification of 4 cell-equivalents per reaction and identified a potential source of MIB in SA-L2 Reservoir. The peak season for benthic growth and secondary metabolite production was observed in spring. Proportionally, 35% of the variability in water geosmin concentrations can be explained by benthic actinobacterial and cyanobacterial activity, showing that freshwater benthic mats represent a significant source of taste and odour compounds.

Deriving safe short-term chemical exposure values (STEV) for drinking water.

Small and brief exceedances of chemicals above their guideline values in drinking water are unlikely to cause an appreciable increased risk to human health. As a result, short-term exposure values (STEV) can be derived to help decide whether drinking water can still be supplied to consumers without adverse health risks. In this study, three approaches were applied to calculate and compare STEV for pesticides. The three approaches included basing a STEV on the acute reference dose (ARfD) (Approach 1), removing conventional attribution rates and uncertainty factors from current guideline values (Approach 2) and extrapolating 1 d and 7 d no observed adverse effect levels (NOAEL) from existing toxicity data using a log-linear regression (Approach 3). Despite being very different methods, the three approaches produced comparable STEV generally within an order of magnitude, which often overlapped with other existing short-term exposure values such as short-term no adverse response levels (SNARL) and health advisories (HA). The results show that adjusting the current guideline value using standard extrapolation factors (Approach 2) often produced the most conservative values. Approach 2 was then applied to two other chemical classes, disinfection by-products (DBPs) and cyanotoxins, demonstrating the wider applicability of the approach.

Transformation of endocrine disrupting chemicals, pharmaceutical and personal care products during drinking water disinfection.

Pharmaceuticals and personal care products (PPCPs) and endocrine disrupting compounds (EDCs) are frequently detected in drinking water sources. This raises concerns about the formation of potentially more toxic transformation products (TPs) after drinking water disinfection. This study applied a combination of computational and experimental methods to investigate the biological activity of eight EDCs and PPCPs commonly detected in source waters (acetaminophen, bisphenol A, carbamazepine, estrone, 17α-ethinylestradiol, gemfibrozil, naproxen and triclosan) before and after disinfection. Using a Stepped Forced Molecular Dynamics (SFMD) method, we detected 911 unique TPs, 36% of which have been previously reported in the scientific literature. We calculated the likelihood that TPs would cause damage to biomolecules or DNA relative to the parent compound based on lipophilicity and the occurrence of structural alerts, and applied two Quantitative Structure-Activity Relationship (QSAR) tools to predict toxicity via receptor-mediated effects. In parallel, batch experiments were performed with three disinfectants, chlorine, chlorine dioxide and chloramine. After solid-phase extraction, the resulting TP mixtures were analyzed by chemical analysis and a battery of eleven in vitro bioassays covering a variety of endpoints. The laboratory results were in good agreement with the predictions. Overall, the combination of computational and experimental chemistry and toxicity methods used in this study suggest that disinfection of the studied EDCs and PPCPs will produce a large number of TPs, which are unlikely to increase specific toxicity (e.g., endocrine activity), but may result in increased reactive and non-specific toxicity.

Benthic cyanobacteria: A source of cylindrospermopsin and microcystin in Australian drinking water reservoirs.

Cyanobacteria represent a health hazard worldwide due to their production of a range of highly potent toxins in diverse aquatic environments. While planktonic species have been the subject of many investigations in terms of risk assessment, little is known about benthic forms and their impact on water quality or human and animal health. This study aimed to purify isolates from environmental benthic biofilms sampled from three different drinking water reservoirs and to assess their toxin production by using the following methods: Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and quantitative PCR (qPCR). Microscopic observation of the isolates allowed the identification of various filamentous cyanobacterial genera: Anabaena (benthic form), Calothrix and Nostoc from the Nostocales and Geitlerinema, Leptolyngbya, Limnothrix, Lyngbya, Oxynema, Phormidium and Pseudanabaena representing non-heterocystous filamentous cyanobacteria. The Phormidium ambiguum strain AWQC-PHO021 was found to produce 739 ng/mg of dry weight (d/w) of cylindrospermopsin and 107 ng/mg (d/w) of deoxy-cylindrospermopsin. The Nostoc linckia strain AWQC-NOS001 produced 400 ng/mg (d/w) of a microcystin analogue. This is the first report of hepatotoxin production by benthic cyanobacteria in temperate Australian drinking water reservoirs. These findings indicate that water quality monitoring programs need to consider benthic cyanobacteria as a potential source of toxins.

Cyanotoxins: Which detection technique for an optimum risk assessment?

The presence of toxigenic cyanobacteria (blue-green algae) in drinking water reservoirs poses a risk to human and animal health worldwide. Guidelines and health alert levels have been issued in the Australian Drinking Water Guidelines for three major toxins, which are therefore the subject of routine monitoring: microcystin, cylindrospermopsin and saxitoxin. While it is agreed that these toxic compounds should be monitored closely, the routine surveillance of these bioactive chemicals can be done in various ways and deciding which technique to use can therefore be challenging. This study compared several assays available for the detection of these toxins and their producers in environmental samples: microscopy (for identification and enumeration of cyanobacteria), ELISA (Enzyme-Linked ImmunoSorbant Assay), PPIA (Protein phosphatase inhibition assay), PSI (Protein synthesis inhibition), chemical analysis and PCR (Polymerase Chain Reaction). Results showed that there was generally a good correlation between the presence of potentially toxigenic cyanobacteria and the detection of the toxin by ELISA. Nevertheless data suggest that cell numbers and toxin concentrations measured in bioassays do not necessarily correlate and that enumeration of potentially toxic cyanobacteria by microscopy, while commonly used for monitoring and risk assessment, is not the best indicator of real toxin exposure. The concentrations of saxitoxins quantified by ELISA were significantly different than those measured by LC-MS, while results were comparable in both assays for microcystin and cylindrospermopsin. The evaluation of these analytical methods led to the conclusion that there is no "gold standard" technique for the detection of the aforementioned cyanotoxins but that the choice of detection assay depends on cost, practicality, reliability and comparability of results and essentially on the question to be answered, notably on toxin exposure potential.

Extended Low-Dose Exposure to Saxitoxin Inhibits Neurite Outgrowth in Model Neuronal Cells.

The potent neurotoxin saxitoxin (STX) belongs to a group of structurally related analogues produced by both marine and freshwater phytoplankton. The toxins act by blocking voltage-gated sodium channels stopping the inflow of sodium ions and the generation of action potentials. Exposure from marine sources occurs as a result of consuming shellfish which have concentrated the toxins, and freshwater exposure can occur from drinking water although there have been no acute poisonings from the latter source to date. Previously, the majority of research into this group of toxins, collectively known as the paralytic shellfish toxins, has focused on acute exposure resulting in paralytic shellfish poisoning. While acute exposure guidelines exist for both sources, there are no chronic exposure guidelines and there has been minimal research into this pattern of exposure despite the known role of electrical activity in neurogenesis. We aimed to investigate this pattern of exposure and its potential effects on neurodevelopment using model neuronal cells. PC12 and SH-SY5Y cells were exposed to STX (0.25-3 µg/l) for 7 days, after which time they were stained with TRITC-Phalloidin, to observe adverse morphological effects. Cells exposed to STX had a significant decrease (18-85%) in long axonlike projections, instead exhibiting a significant increase in shorter projections classified as filopodia (p < 0.05). The results suggest that extended low-dose exposure to STX can inhibit proper neurite outgrowth at concentrations well below guideline levels for both sources of exposure making it a potential public health concern.

Hypothetical scenario exercises to improve planning and readiness for drinking water quality management during extreme weather events.

Two hypothetical scenario exercises were designed and conducted to reflect the increasingly extreme weather-related challenges faced by water utilities as the global climate changes. The first event was based on an extreme flood scenario. The second scenario involved a combination of weather events, including a wild forest fire ('bushfire') followed by runoff due to significant rainfall. For each scenario, a panel of diverse personnel from water utilities and relevant agencies (e.g. health departments) formed a hypothetical water utility and associated regulatory body to manage water quality following the simulated extreme weather event. A larger audience participated by asking questions and contributing key insights. Participants were confronted with unanticipated developments as the simulated scenarios unfolded, introduced by a facilitator. Participants were presented with information that may have challenged their conventional experiences regarding operational procedures in order to identify limitations in current procedures, assumptions, and readily available information. The process worked toward the identification of a list of specific key lessons for each event. At the conclusion of each simulation a facilitated discussion was used to establish key lessons of value to water utilities in preparing them for similar future extreme events.

Low dose extended exposure to saxitoxin and its potential neurodevelopmental effects: A review.

Saxitoxin (STX) and its analogs, the paralytic shellfish toxins (PSTs), are a group of potent neurotoxins well known for their role in acute paralytic poisoning by preventing the generation of action potentials in neuronal cells. They are found in both marine and freshwater environments globally and although acute exposure from the former has previously received more attention, low dose extended exposure from both sources is possible and to date has not been investigated. Given the known role of cellular electrical activity in neurodevelopment this pattern of exposure may be a significant public health concern. Additionally, the presence of PSTs is likely to be an ongoing and possibly increasing problem in the future. This review examines the neurodevelopmental toxicity of STX, the risk of extended or repeated exposure to doses with neurodevelopmental effects, the potential implications of this exposure and briefly, the steps taken and difficulties faced in preventing exposure.

Effect of increasing bromide concentration on toxicity in treated drinking water.

Research is increasingly indicating the potential chronic health effects of brominated disinfection by-products (DBPs). This is likely to increase with elevated bromide concentrations resulting from the impacts of climate change, projected to include extended periods of drought and the sudden onset of water quality changes. This will demand more rigorous monitoring throughout distribution systems and improved water quality management at water treatment plants (WTPs). In this work the impact of increased bromide concentration on formation of DBPs following conventional treatment and chlorination was assessed for two water sources. Bioanalytical tests were utilised to determine cytotoxicity of the water post disinfection. Coagulation was shown to significantly reduce the cytotoxicity of the water, indicating that removal of natural organic matter DBP precursors continues to be an important factor in drinking water treatment. Most toxic species appear to form within the first half hour following disinfectant addition. Increasing bromide concentration across the two waters was shown to increase the formation of trihalomethanes and shifted the haloacetic acid species distribution from chlorinated to those with greater bromine substitution. This correlated with increasing cytotoxicity. This work demonstrates the challenges faced by WTPs and the possible effects increasing levels of bromide in source waters could have on public health.

Undifferentiated murine embryonic stem cells used to model the effects of the blue-green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation.

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 µg/L. mES exposed to 0-1 µg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 µg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 µg/mL, the Lowest OEC was 1 µg/mL (p < 0.001, n = 3), and the IC50 was 0.86 µg/mL after 24 h. The ADWG 1 µg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins.

Evaluating Evidence for Association of Human Bladder Cancer with Drinking-Water Chlorination Disinfection By-Products.

Exposure to chlorination disinfection by-products (CxDBPs) is prevalent in populations using chlorination-based methods to disinfect public water supplies. Multifaceted research has been directed for decades to identify, characterize, and understand the toxicology of these compounds, control and minimize their formation, and conduct epidemiologic studies related to exposure. Urinary bladder cancer has been the health risk most consistently associated with CxDBPs in epidemiologic studies. An international workshop was held to (1) discuss the qualitative strengths and limitations that inform the association between bladder cancer and CxDBPs in the context of possible causation, (2) identify knowledge gaps for this topic in relation to chlorine/chloramine-based disinfection practice(s) in the United States, and (3) assess the evidence for informing risk management. Epidemiological evidence linking exposures to CxDBPs in drinking water to human bladder cancer risk provides insight into causality. However, because of imprecise, inaccurate, or incomplete estimation of CxDBPs levels in epidemiologic studies, translation from hazard identification directly to risk management and regulatory policy for CxDBPs can be challenging. Quantitative risk estimates derived from toxicological risk assessment for CxDBPs currently cannot be reconciled with those from epidemiologic studies, notwithstanding the complexities involved, making regulatory interpretation difficult. Evidence presented here has both strengths and limitations that require additional studies to resolve and improve the understanding of exposure response relationships. Replication of epidemiologic findings in independent populations with further elaboration of exposure assessment is needed to strengthen the knowledge base needed to better inform effective regulatory approaches.

Extreme weather events: Should drinking water quality management systems adapt to changing risk profiles?

Among the most widely predicted and accepted consequences of global climate change are increases in both the frequency and severity of a variety of extreme weather events. Such weather events include heavy rainfall and floods, cyclones, droughts, heatwaves, extreme cold, and wildfires, each of which can potentially impact drinking water quality by affecting water catchments, storage reservoirs, the performance of water treatment processes or the integrity of distribution systems. Drinking water guidelines, such as the Australian Drinking Water Guidelines and the World Health Organization Guidelines for Drinking-water Quality, provide guidance for the safe management of drinking water. These documents present principles and strategies for managing risks that may be posed to drinking water quality. While these principles and strategies are applicable to all types of water quality risks, very little specific attention has been paid to the management of extreme weather events. We present a review of recent literature on water quality impacts of extreme weather events and consider practical opportunities for improved guidance for water managers. We conclude that there is a case for an enhanced focus on the management of water quality impacts from extreme weather events in future revisions of water quality guidance documents.

Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water.

The growing use of recycled water in large urban centres requires comprehensive public health risk assessment and management, an important aspect of which is the assessment and management of residual trace chemical substances. Bioanalytical methods such as in vitro bioassays may be ideal screening tools that can detect a wide range of contaminants based on their biological effect. In this study, we applied thirteen in vitro assays selected explicitly for their ability to detect molecular and cellular effects relevant to potential chemical exposure via drinking water as a means of screening for chemical contaminants from recycled water at 9 Australian water reclamation plants, in parallel to more targeted direct chemical analysis of 39 priority compounds. The selected assays provided measures of primary non-specific (cytotoxicity to various cell types), specific (inhibition of acetylcholinesterase and endocrine receptor-mediated effects) and reactive toxicity (mutagenicity and genotoxicity), as well as markers of adaptive stress response (modulation of cytokine production) and xenobiotic metabolism (liver enzyme induction). Chemical and bioassay analyses were in agreement and complementary to each other: the results show that source water (treated wastewater) contained high levels of biologically active compounds, with positive results in almost all bioassays. The quality of the product water (reclaimed water) was only marginally better after ultrafiltration or dissolved air floatation/filtration, but greatly improved after reverse osmosis often reducing biological activity to below detection limit. The bioassays were able to detect activity at concentrations below current chemical method detection limits and provided a sum measure of all biologically active compounds for that bioassay, thus providing an additional degree of confidence in water quality.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems.

Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin--marine biotoxins--poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse as well as the rat bioassay. Besides ethical concerns, there is also a need for more reliable test methods because of low inter-species comparability, high intra-species variability, the high number of false positive and negative results as well as questionable extrapolation of quantitative risk to humans. For this reason, a transatlantic group of experts in the field of marine biotoxins was convened from academia and regulatory safety authorities to discuss future approaches to marine biotoxin testing. In this report they provide a background on the toxin classes, on their chemical characterization, the epidemiology, on risk assessment and management, as well as on their assumed mode of action. Most importantly, physiological functional assays such as in vitro bioassays and also analytical techniques, e.g., liquid chromatography coupled mass spectrometry (LC-MS), as substitutes for the rodent bioassay are reviewed. This forms the basis for recommendations on methodologies for hazard monitoring and risk assessment, establishment of causality of intoxications in human cases, a roadmap for research and development of human-relevant functional assays, as well as new approaches for a consumer directed safety concept.

Health risk assessment for cyanobacterial toxins in seafood.

Cyanobacteria (blue-green algae) are abundant in fresh, brackish and marine waters worldwide. When toxins produced by cyanobacteria are present in the aquatic environment, seafood harvested from these waters may present a health hazard to consumers. Toxicity hazards from seafood have been internationally recognised when the source is from marine algae (dinoflagellates and diatoms), but to date few risk assessments for cyanobacterial toxins in seafood have been presented. This paper estimates risk from seafood contaminated by cyanobacterial toxins, and provides guidelines for safe human consumption.

Cylindrospermopsin, a blue-green algal toxin, inhibited human luteinised granulosa cell protein synthesis in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) inhibits protein synthesis, and CYP450 enzymes metabolise CYN to cytotoxic endproducts. Human chorionic gonadotrophin (hCG) stimulates the de novo synthesis of StAR and CYP450 aromatase. Human IVF-derived granulosa cells (GC) (n=7) were exposed to 0-5µM CYN±1IU/ml hCG for 2-24h. After 24h pre-culture GC responded to hCG by increasing estradiol 17ß (E(2)) and progesterone (P(4)) synthesis. Three micromolar of CYN±1IU/ml hCG for 24h was not cytotoxic and did not affect basal or hCG-stimulated E(2) or P(4) production, but did inhibit protein synthesis (p<0.05, n=4). hCG-stimulated steroidogenesis was not reduced by CYN, suggesting a lack of effect on StAR or CYP450 aromatase protein synthesis. hCG enhanced the effects of CYN on GC protein synthesis. Twenty four hours exposure to 0.1µM CYN did not affect GC, supporting the establishment of a 0.0024µM Guideline level for CYN in public water supplies.

Toxicity of the cyanobacterium Limnothrix AC0243 to male Balb/c mice.

A growing list of freshwater cyanobacteria are known to produce toxic agents, a fact which makes these organisms of concern to water authorities. A cultured strain of Limnothrix (AC0243) was recently shown to have toxic effects in in vitro bioassays. It did not produce any of the known cyanobacterial toxins. The intrapertoneal toxicity of aqueous extracts of the material was therefore tested in mice to determine whether the observed effects might be of public health relevance to drinking water supplies. The results indicate that Limnothrix AC0243 is acutely toxic to mice, causing widespread cellular necrosis in the liver, kidneys and gastrointestinal tract within 24 h of exposure. Sub-lethal effects lasted at least 7 d. These results suggest that Limnothrix AC0243 produces a novel toxin ("Limnothrixin") and that further work is therefore urgently required to quantify the potential public health implications.

Community composition, toxigenicity, and environmental conditions during a cyanobacterial bloom occurring along 1,100 kilometers of the Murray River.

A cyanobacterial bloom impacted over 1,100 km of the Murray River, Australia, and its tributaries in 2009. Physicochemical conditions in the river were optimal to support a bloom at the time. The data suggest that at least three blooms occurred concurrently in different sections of the river, with each having a different community composition and associated cyanotoxin profile. Microscopic and genetic analyses suggested the presence of potentially toxic Anabaena circinalis, Microcystis flos-aquae, and Cylindrospermopsis raciborskii at many locations. Low concentrations of saxitoxins and cylindrospermopsin were detected in Anabaena and Cylindrospermopsis populations. A multiplex quantitative PCR was used, employing novel oligonucleotide primers and fluorescent TaqMan probes, to examine bloom toxigenicity. This single reaction method identified the presence of the major cyanotoxin-producing species present in these environmental samples and also quantified the various toxin biosynthesis genes. A large number of cells present throughout the bloom were not potential toxin producers or were present in numbers below the limit of detection of the assay and therefore not an immediate health risk. Potential toxin-producing cells, possessing the cylindrospermopsin biosynthesis gene (cyrA), predominated early in the bloom, while those possessing the saxitoxin biosynthesis gene (sxtA) were more common toward its decline. In this study, the concentrations of cyanotoxins measured via enzyme-linked immunosorbent assay (ELISA) correlated positively with the respective toxin gene copy numbers, indicating that the molecular method may be used as a proxy for bloom risk assessment.

Cytotoxic and genotoxic effects of cylindrospermopsin in mice treated by gavage or intraperitoneal injection.

Cylindrospermopsin (CYN), a cyanobacterial hepatotoxin mainly produced by Cylindrospermopsis raciborskii, has been involved in human intoxications and livestock deaths. The widespread occurrence of CYN in the water supplies lead us to investigate its genotoxicity to assess potential chronic effects. This study reports evaluation of CYN-induced in vivo DNA damage in mice using alkaline comet assay (ACA) and micronucleus assay (MNA) concomittantly. ACA measures DNA breakage from single and double strand breaks as well as alkali labile sites. Conversely, MNA detects chromosome damage events such as chromosomal breakage and numeric alterations. Male Swiss mice were treated with CYN concentrations of 50, 100, and 200 µg/kg by a single intraperitoneal (ip) injection or with 1, 2, and 4 mg/kg by gavage. Methyl methane sulfonate (MMS) was used as positive control at 80 mg/kg. Twenty-four hours after treatment, samples of liver, blood, bone marrow, kidney, intestine, and colon were taken to perform ACA, the bone marrow and the colon were also used for MNA. Parameters used to quantify DNA damage were % Tail DNA for ACA and both micronucleated immature erythrocytes and epithelial colon cells for MNA. DNA breaks and chromosome damage were significantly increased by MMS in all the organs evaluated. Significant DNA damage was detected within the colon by ACA after ip injection of 100 and 200 µg/kg CYN (P < 0.01). DNA damage was also detected in colon samples after 4 mg/kg oral administration of CYN and in bone marrow after 1 and 2 mg/kg of orally administered CYN. Histological examination showed foci of cell death within the liver and the kidney from mice that received the two highest doses of CYN by either route of administration.