Joseph A. Cotruvo

Therapeutic dose as the point of departure in assessing potential health hazards from drugs in drinking water and recycled municipal wastewater

The detection of drugs in drinking water sources has raised questions related to safety. In the absence of regulatory or other official guidance, water utilities are faced with a problem of which drugs should be monitored and the detection limits that should be required. The US FDA summarizes data required for drug approval and post marketing adverse reaction reporting. The use of these data as a means of arriving at concentrations in water where adverse health effects are minimal or non-existent was explored. The minimum therapeutic dose was assumed an appropriate point of departure. Appropriate uncertainty factors could be applied depending upon the qualitative and quantitative nature of the data that are available. Assumptions inherent in US FDAs approval of drugs for use in subsets of the population relative to the broader concerns that arise for exposures of the entire population had to be considered. Additional questions are; whether the drug under consideration is carcinogenic, carries pregnancy and lactation warnings, approval for limited vs. chronic use, exposures to multiple compounds that could act in additive or synergistic ways, and the seriousness of toxicities that are observed. Aside from these considerations, a combined uncertainty factor of 1000 appeared adequate.

Cellular and molecular mechanisms of bromate-induced cytotoxicity in human and rat kidney cells

The mechanisms of bromate (BrO(3)(-))-induced toxicity in Normal Rat Kidney (NRK) and human embryonic kidney 293 (HEK293) cells were investigated. BrO(3)(-) (added as KBrO(3)) induced concentration-dependent decreases in 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) staining after 48 h. BrO(3)(-)-induced necrosis based on tandem increases in annexin V and PI staining. Cell cycle analysis demonstrated that BrO(3)(-) also induced G2/M arrest and nuclear fragmentation, prior to alterations in MTT staining or annexin V and PI staining. Immunoblot analysis demonstrated that the G2/M arrest correlated to induction of phosphorylated (p)-p53, p21, cyclin B1 and p-cdc2. Further, BrO(3)(-) induced time-dependent increases in the activity of the mitogen activated protein kinases p38 and ERK1/2. Treatment of cells with the p38 inhibitor SB202190, but not the ERK1/2 inhibitor PD98059, partially reversed BrO(3)(-)-induced G2/M arrest and decreased BrO(3)(-)-induced p-p53, p21 and cyclin B1 expression. In addition, BrO(3)(-) treatment induced reactive oxygen species (ROS) based on increases in CM-H(2)DCFDA fluorescence. The antioxidant ascorbic acid inhibited BrO(3)(-)-induced p38 activation, G2/M arrest, p-p53, p21 and cyclin B1 expression; however, ascorbic acid had no effect on BrO(3)(-)-induced formation of 8-OHdG, a marker of DNA oxidative damage, whose increases preceded cell death by 24h. These data suggest that ROS mediated MAPK activation is involved in the molecular mechanisms of BrO(3)(-)-induced cell cycle arrest, which occurs independently of 8-OH-dG production. The similar mode of action in both NRK and HEK293 cells suggests that the mechanisms of BrO(3)(-)-induced renal cell death are model-independent.

Risk assessment of formaldehyde

Abstract In April 1987, the Environmental Protection Agency (EPA) issued the “Assessment of Health Risks to Garment Workers and Certain Home Residents from Exposure to Formaldehyde” in which formaldehyde was classified as a carcinogen and an irritant to the eyes and respiratory tract. A quantitative risk assessment for cancer was presented. A more current document, a draft released in 1991, incorporates some additional data on the epidemiology and toxicology of formaldehyde that the EPA has received since completion of the earlier assessment, and examines the impact of this information on the estimates of health risks following exposure to airborne formaldehyde. For noncancer effects, the new data support earlier conclusions with regard to the irritant effects of formaldehyde and the dose-response gradient for these effects. The cancer assessment incorporates the use of a molecular dosimeter for the derivation of risk estimates. Tissue levels of this dosimeter, a covalent cross-link product of formaldehyde and DNA-protein (DPX), are available from rats and monkeys. The risk estimates obtained with this dosimeter are considerably lower than those obtained by conventional approaches.

Organic micropollutants in drinking water: an overview.

Biological contamination is still the most significant public health risk from drinking water even in industrialized countries. High potential for organic chemical transport to drinking water continues to exist even with source protection because of the multitude of chemical types and quantities. Drinking water is usually not a unique source nor the most significant contributor to total exposure from synthetic organic chemicals but it might be one of the most controllable. The major public concern with drinking water contamination has been possible contribution to cancer risks from organic micropollutants. Even though the actual risks are probably small in most cases it is clearly within the public interest to prevent adulteration of water supplies and to protect their quality for the future so that these concerns or risks can be avoided. A risk assessment/management decision model is suggested which may assist the process of making rational assessments of these contamination problems and control decisions that consciously consider all of the available data in a consistent manner.

The synergistic effect of sodium chlorite and bromochloroacetic acid on BrO3−-induced renal cell death

Bromate (BrO(3)(-)) is a drinking water disinfection by-product (DBP) that induces renal cell death via DNA damage-dependent and -independent mechanisms. Drinking water contains other DBPs in addition to BrO(3)(-). We tested the effect of two of these, sodium chlorite (NaClO(2)) and bromochloroacetic acid (BCAA), on BrO(3)(-) cytotoxicity in normal rat kidney (NRK) cells. NaClO(2) and BCAA alone induced cytotoxicity at concentrations of over 20ppm, while BrO(3)(-) was only moderately cytotoxic at concentrations of 200ppm. Combining BrO(3)(-) with NaClO(2) or BCAA alone enhanced cytotoxicity 1.5-4 fold. Exposing cells to all three compounds induced synergistic-like increases in cytotoxicity. This effect did not correlate to increases in reactive oxygen species (ROS), even though all three compounds induced ROS formation alone. NaClO(2), but not BCAA, increased BrO(3)(-)-mediated DNA damage as measured by 8-hydroxydeoxyguanosine (8-OHdG) staining. In addition, NaClO(2), but not BCAA, decreased BrO(3)(-)-induced G2/M cell cycle arrest. Both compounds increased apoptosis in the presence of BrO(3)(-) as assessed by annexin V, PI, and DAPI staining. This is in contrast to BrO(3)(-) treatment alone, which induced necrosis. Immunoblot analysis showed that both NaClO(2) and BCAA increased p38 activation; however, consistent with 8-OHdG staining, only NaClO(2) increased BrO(3)(-)-induced histone H2AX phosphorylation, a marker of DNA damage. In contrast, BCAA, but not NaClO(2), increased BrO(3)(-)-induced phosphorylation of p53. These data support the novel finding that mixtures of DBPs increase BrO(3)(-)-induced renal cell death by DNA-dependent and -independent mechanisms, and could alter how the risk of these DBPs towards humans is assessed.

Drinking water as a proportion of total human exposure to volatile N-nitrosamines.

Some volatile N-nitrosamines, primarily N-nitrosodimethylamine (NDMA), are recognized as products of drinking water treatment at ng/L levels and as known carcinogens. The U.S. EPA has identified the N-nitrosamines as contaminants being considered for regulation as a group under the Safe Drinking Water Act. Nitrosamines are common dietary components, and a major database (over 18,000 drinking water samples) has recently been created under the Unregulated Contaminant Monitoring Rule. A Monte Carlo modeling analysis in 2007 found that drinking water contributed less than 2.8% of ingested NDMA and less than 0.02% of total NDMA exposure when estimated endogenous formation was considered. Our analysis, based upon human blood concentrations, indicates that endogenous NDMA production is larger than expected. The blood-based estimates are within the range that would be calculated from estimates based on daily urinary NDMA excretion and an estimate based on methylated guanine in DNA of lymphocytes from human volunteers. Our analysis of ingested NDMA from food and water based on Monte Carlo modeling with more complete data input shows that drinking water contributes a mean proportion of the lifetime average daily NDMA dose ranging from between 0.0002% and 0.001% for surface water systems using free chlorine or between 0.001% and 0.01% for surface water systems using chloramines. The proportions of average daily dose are higher for infants (zero to six months) than other age cohorts, with the highest mean up to 0.09% (upper 95th percentile of 0.3%).

Association of brominated proteins and changes in protein expression in the rat kidney with subcarcinogenic to carcinogenic doses of bromate.

The water disinfection byproduct bromate (BrO3(-)) produces cytotoxic and carcinogenic effects in rat kidneys. Our previous studies demonstrated that BrO3(-) caused sex-dependent differences in renal gene and protein expression in rats and the elimination of brominated organic carbon in their urine. The present study examined changes in renal cell apoptosis and protein expression in male and female F344 rats treated with BrO3(-) and associated these changes with accumulation of 3-bromotyrosine (3-BT)-modified proteins. Rats were treated with 0, 11.5, 46 and 308 mg/L BrO3(-) in drinking water for 28 days and renal sections were prepared and examined for apoptosis (TUNEL-staining), 8-oxo-deoxyguanosine (8-oxoG), 3-BT, osteopontin, Kim-1, clusterin, and p-21 expression. TUNEL-staining in renal proximal tubules increased in a dose-related manner beginning at 11.5mg BrO3(-)/L in female rats and 46 mg/L in males. Increased 8-oxoG staining was observed at doses as low as 46 mg/L. Osteopontin expression also increased in a dose-related manner after treatment with 46 mg/L, in males only. In contrast, Kim-1 expression increased in a dose-related manner in both sexes, although to a greater extent in females at the highest dose. Clusterin and p21 expression also increased in a dose-related manner in both sexes. The expression of 3-BT-modified proteins only increased in male rats, following a pattern previously reported for accumulation of α-2u-globulin. Increases in apoptosis in renal proximal tubules of male and female rats at the lowest doses suggest a common mode of action for renal carcinogenesis for the two sexes that is independent of α-2u-globulin nephropathy.

EPA Policies to Protect the Health of Consumers of Drinking Water in the United States

In the United States our objective is to protect drinking water at the source, during treatment and during distribution. In 1975 interim regulations for bacteria and turbidity, 10 inorganic chemicals, 6 organic chemicals and radionuclides were promulgated. In 1979 National Secondary Regulations for substances affecting the aesthetic quality of water were promulgated. In 1979 trihalomethanes were added. The United States is engaged in comprehensive revisions of the National Primary Drinking Water Regulations. The areas of our most significant concern include detection and control of contamination of ground waters by organic chemicals resulting from improper waste disposal practices, a reassessment of microbiological regulations and toxicity of disinfectant by-products and a major effort to deal with corrosion-related contamination of drinking water during distribution. We are evaluating the issue of a granular activated carbon requirement for contaminated surface waters. A program to assure the quality of direct and indirect additives to drinking water has also been initiated. Part of this activity will include determination of the contaminants and by-products associated with the use of various water treatment chemicals and pipe materials.

Nongenotoxic Mechanisms Involved in Bromate-Induced Cancer in Rats

Risk assessments for bromate (BrO3-) in drinking water are based on linear extrapolation from the total incidence of tumors in male rats. The only genotoxic effects that might result from carcinogenic doses of BrO3- in vivo are the formation of 8-oxodeoxyguanosine (8-oxoG) in deoxyribonucleic acid (DNA) and the production of micronuclei. The mutations in tumors are consistent with the 8-oxoG adduct, and both effects are nonlinear with respect to dose. Treatment of rats with BrO3- resulted in bromination of protein tyrosines. The accumulation of these proteins in the kidney appeared to contribute to kidney cancer in male, but not female, rats. BrO3- increased the rate of apoptosis (programmed cell death) in the kidneys of rats of both sexes, an effect associated with increasing expression of antiapoptotic genes and proteins. Consequently, suppression of apoptosis is a likely mechanism for BrO3- induced kidney cancer. More limited data suggest nongenotoxic modes of action for thyroid tumors and mesotheliomas. If these data are confirmed, linear extrapolation of risk to low doses is inappropriate.

Evaluation of Point of Use (POU) Systems for the Removal of Microbiological Contaminants in Drinking Water

As a final barrier before consumption, properly designed and operated Point-of-Use (POU) treatment systems are important for safe drinking water supplies. POU treatment devices have gained importance for protection against intentional or accidental contamination of drinking water supplies. The events of September 11, 2001 have further emphasized the importance of POU devices in this regard. This paper summarizes the results of pilot-scale studies that were conducted at the U.S. Environmental Protection Agency’s (EPA’s) Test and Evaluation (T&E) Facility in Cincinnati, Ohio, to evaluate the performance of three different POU systems for removing microbiological contaminants in drinking water. Two of these systems were based on filtration through electrostatically charged media and the third system incorporated a reverse osmosis (RO) membrane. The systems were challenged with a number of microbiological contaminants including Cryptosporidium, E. coli, MS2 bacteriophage, B. subtilis and polystyrene latex (PSL) beads (as a surrogate for Cryptosporidium). The results demonstrated that these POU systems were effective in removing the target biological contaminants in accordance with the standards presented in the U.S. EPA “Guide Standard and Protocol for Testing Microbiological Water Purifiers” (USEPA, 1987).