Source: KENTUCKY STATE UNIVERSITY submitted to
PROTECTIVE EFFECTS OF PHYTOCHEMICALS AGAINST OXIDATIVE CHANGES INDUCED BY PESTICIDE EXPOSURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0196016
Grant No.
(N/A)
Project No.
KYX-50-03-17H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2003
Project End Date
Sep 30, 2007
Grant Year
(N/A)
Project Director
Panemangalore, M.
Recipient Organization
KENTUCKY STATE UNIVERSITY
(N/A)
FRANKFORT,KY 40601
Performing Department
Human Nutrition & Health
Non Technical Summary
This study will assess the protective effects of phytochemicals against the harmful changes induced by exposure to pesticides. The goal of this research proposal is to identify the protective effects of phytochemicals on the induction of oxidative stress upon exposure to commonly used pesticides in Hep G2 human liver cells. Since human hepatocytes will be used the data could be more readily extrapolated to humans.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7027010101060%
7027010115040%
Goals / Objectives
1 Determine the changes in cytochrome P450 and endogenous antioxidants in Hep G2 cells after exposure to pesticides and flavonoids; establish the development of oxidative stress. 2 Assess the protective effects of flavonoids and carotenoids in Hep G2 cells after exposure to pesticides either singly or in combination on cytochrome P450 (CYP 450) enzymes and endogenous antioxidants 3 Evaluate the protective effects of phytochemicals and carotenoids on DNA damage induced by pesticide exposure in Hep G2 cells
Project Methods
Objective1: First we will establish Hep G2 cell culturing in our lab using Dulbecco's MEM and 10% FBS and determine the need to add selenium to the culture medium to ensure normal or stable glutathione peroxidase activity (GPX) in Hep G2 cells. The activities or concentrations of cytochrome P450 1A (CYP1A), superoxide dismutase (SOD), GPX and glutathione (GSH) at confluence will also be determined in these cells. In addition, we will assess the cytotoxicity (NI50) of the pesticides and flavonoids in Hep G2 cells using neutral red. The effects of different concentrations of mixtures of pesticides efsenvalerate (organophosphate), carbaryl (carbamate) and endosulfan (organochlorine), and incubation time on changes in CYP 1A and the endogenous antioxidants will be studied to determine time and concentration response. Similarly, the effects of flavonoids quercetin (Qu), rutin (Ru), catechin (Ce) and kaempherol (Ka) on CYP 1A and the endogenous antioxidants will be assessed both individually or in combination. Objective 2: The protective effects of flavonoids and carotenoids (lutein and lycopene) added before, after or together with the concentration of the pesticide mixtures that induces ROS will be determined; for comparison, the protective effects of flavonoids and carotenoids using hydrogen peroxide will also be ascertained using the protocols standardized previously. We will also assess the extent of oxidative stress in the cells after various treatments using ethidium fluorescence produced after oxidation of dihydroethidium as a specific indicator of free radical oxygen. The role of GSH and CYP 3A in ROS metabolism under our experimental conditions will be assessed using specific inhibitors. Objective 3: The potential beneficial effects of flavonoids and carotenoids in modulating the genotoxic effects of pesticide exposure will be determined in these experiments. We will initially determine the concentration and type of pesticide mixture that produces DNA damage in Hep G2 cells. DNA damage will be assessed by the modified comet assay that detects oxidized DNA bases. The effectiveness of flavonoids or carotenoids added before and after development of DNA damage or with the DNA damaging dose of pesticide mixture will be tested in Hep G2 cells. The exposure protocol and single cell electrophoresis (comet assay) methodology will be standardized for these studies. The statistical experimental design for all studies will be chosen based on the objective and the experimental protocol to determine the significant differences in the data and assess the effects of flavonoids.

Progress 07/01/03 to 09/30/07

Outputs
OUTPUTS: The goals of this project were to ascertain the protective effects of flavonoids in presence of pesticide exposure and also to assess their metabolic effects at varied levels of intake. The two objectives were to determine: 1) the effect of administering different levels of a purified flavonoid mixture (F) as a bolus and 2) the addition of F in the diet with concomitant exposure to a low level pesticide mixture (P) on changes in the concentrations of zinc and copper and superoxide dismutase (SOD) activity in tissues of rats. Male SD rats (150-175g, 6/group/both experiments (EX)) had free access to the AIN-93 diet and distilled water. EX1: a mixture of equal amounts of F (quercetin (Q), rutin (R) and catechin (C)) at 5, 25, 50, 100 and 200 mg/kg bw (Groups: CON, F5, F25, F50, F100, F200) was dissolved in PEG 100 and fed by gavage (0.5 mL/day/5days a wk; 0.5 mL PEG alone for CON group) for 4 weeks. EX2: mixture of Q, R and C was added at 1 or 5 mM/kg diet (AIN93) and P mixture: endosulfan + Thiram + Chlorpyrifos in oil was gavage fed 5 days a week at 2.5% of LD50 (Groups: CON, P, F1, F 5, P+F1, P+F5) for 4 weeks. Results: EX1: Mean F intake ranged from for F5 = 1 mg/day to F200 = 46 mg/day; Plasma: Cu decreased by about 25%-30% in all F groups without a concomitant decrease in ceruloplasmin activity compared to CON; Liver: Cu declined by about 20% in all FM groups; Small Intestines (SI) Mucosa: Cu retention decreased by 17% and 26% in F100 and F200 groups compared to CON, respectively, (P≤0.05). SOD activities: Erythrocyte SOD was unchanged; Liver: SOD was reduced in groups fed F50, F100 or F200 by 28%, 35% and 48% compared to CON, respectively; SI Mucosa: SOD increased by 40% in groups F25, F50 and F100 and decreased 20% in FM200 compared to the CON (P≤0.05). EX2: F consumed from the diet was about 20 and 100 mg/day for F1 and F5 groups, respectively; Plasma: no changes in Zn were found and increase in Cu ranged from 16% to 36% more than the CON in all P and F groups; Liver: Zn and Cu levels were similar to the CON; SI Mucosa: Zn retention increased by 20% in all P and F groups; Cu retention increased by 46% in P and F1, F5 groups and by 38% in the P+F groups above the CON (P≤0.05). SOD activities: Erythrocyte and SI Mucosa SOD was unchanged; liver SOD was 50% higher in all groups compared to the CON. These disparate changes in plasma and tissue Zn and Cu concentrations could be attributed to the route of administration and level of flavonoid intake, which was higher in EX2 than EX1. The changes in SOD activity were probably related to either inhibition or activation by flavonoids and/or their metabolites. The interactive effects between P and F were minimal. Clearly, feeding of purified flavonoids as a bolus than as an integral part of the diet could compromise Cu and to some extent Zn homeostasis in tissues. PARTICIPANTS: Frederick N Bebe. Co-Investigator. Mr. Bebe had the opportunity to particpate in national meetings to present the research conducted on this project and attend symposia and colloquia at these professional meetings. TARGET AUDIENCES: Dieticians, nutritionists and extension personnel involved in counseling and improving food choices and eating habits of various populations.

Impacts
These studies underscore the importance of consuming flavonoids from foods rather than as nutraceuticals (tablets or capsules) to avoid compromising copper homeostasis. As most US population groups obtain marginal levels of copper from their diet, it is important to emphasize the importance of consuming fruits and vegetables that are rich in flavonoids to obtain the maximum advantage of these bioactive compounds. Also, though these compounds are metabolized by the same pathway as pesticides and other environmental pollutants, their additive metabolic effects appear to be minimal.

Publications

  • Panemangalore M and Bebe FN (2007). Effects of Flavonoids on Endogenous Antioxidants in Tissues of Rats Exposed to Low Levels of Pesticides. FASEB J 21: Abstract # 700.6. Bebe FN and Panemangalore M (2007). The Effect of Flavonoids on Pesticide Induced Decline in Trace Mineral Concentrations in Tissues of Rats. FASEB J 21: Abstract # 698.9.


Progress 01/01/06 to 12/31/06

Outputs
Fruits and vegetables contain naturally occurring secondary metabolites such as flavonoids, carotenoids and glucosinolates that have significant beneficial biological effects. Many flavonoids have been shown to possess antioxidant and free radical scavenging properties and have been reported to reduce oxidative stress in cell culture studies. By contrast, pesticides have been shown to induce oxidative stress in animals and humans by compromising endogenous antioxidants. The goals of this project are to ascertain the protective effects of flavonoids in presence of pesticide exposure and also to assess their biological safety at high levels. In the first experiment the protective potential of flavonoids to modify the effects of pesticides was studied in rats. Male SD rats, weighing 150-175g, 6/group were fed AIN-93 diet (CON) or 1.0 mM/kg diet of a mixture of flavonoids (FM): quercetin, rutin and catechin. Pesticide mixture (PM) containing acephate, endosulfan and carbaryl at 25% LD50 in oil was fed orally at 0.1mL/5d/week. Changes in Cu concentrations were significant: PM and FM alone reduced plasma Cu by 15% and 17%, respectively; combined exposure to PM + FM decreased plasma Cu by 43% (P≤0.05). The reduction in plasma Cu was accompanied by a concomitant decline in plasma ceruloplasmin (CP) diamine esterase activity (P≤0.05). Intestinal mucosa (IM) Cu concentrations declined by an average of 38%, 22% and 37%, respectively, in the PM, FM and PM + FM groups; a 31% reduction was found in IM Zn concentrations of all groups. FM increased IM SOD activity by 25%, while FM+PM decreased SOD by 26%. In the liver, glutathione peroxidase (GPX) activity was reduced by 16% , 22% and 25% in the PM, FM and FM + PM groups, respectively, whereas, FM alone increased GPX activity in the IM by 31% and maintained normal GPX activity in the PM + FM group (P≤0.05). This study suggests that the flavonoid mixture largely protected decline in mucosal GPX activity induced by pesticides exposure. However, the concordant decrease in plasma Cu and ceruloplasmin (Cu carrier protein) suggests a compromise of Cu status in animals exposed to PM, FM or PM + FM. Thus, it is possible that the ingestion of flavonoids under conditions of low level pesticide exposure could further compromise copper status. A similar study is in progress to determine the effects of long term exposure to PM and FM at different levels in the diet. In another planned experiment we will be assessing the effects of feeding graded doses of flavonoid mixtures on endogenous antioxidants, and zinc and copper status.

Impacts
The consumption of flavonoids has increased because of their much vaunted protective properties. Their easy access and availability at "nutrition" and other health food stores, which sell combinations of flavonoids and other bioactive compounds at pharmaceutical concentrations and are often called neutraceuticals has lead to increased consumption by the public. Moreover, these products are not currently regulated by the FDA and additional data on biosafety are essential. The results from this project will provide evidence on the in vivo biosafety of these phytochemicals and elaborate on their protective effects. Most of all, the data would be specifically applicable to rural population groups that are either occupationally or environmentally exposed to pesticides.

Publications

  • Tope AM and Panemangalore M. (2007). Assessment of Oxidative Stress Due to Exposure to Pesticides in Plasma and Urine of Traditional Limited-Resource Farm Workers: Formation of the DNA-Adduct 8-hydroxy-2-deoxy-guanosine (8-OHdG). J Environ Sci Hlth Part B, in press.
  • Bebe FN and Panemangalore M. (2006). Pesticides in the diet modify trace metal homeostasis in rats. Association of Research Directors, Inc., 14th Biennial Research Symposium Abstracts: Abstract # P160, page 257.
  • Panemangalore M, Tope AM and Bebe FN. (2006). Worker Pesticide Exposure. Published online in the Encyclopedia of Pest Management. Editor: Pimental D. Francis and Taylor, New York. Pages 1-3.
  • Tope AM, Bebe FN and Panemangalore M. (2006). Increased micronuclei formation in lymphocytes and modification of blood antioxidants in farm workers during the growing season. J Environ Sci Hlth Part B. 41, Part B: 843-853.


Progress 01/01/05 to 12/31/05

Outputs
Previous research has shown that dermal, oral or whole body exposure to low levels of mixtures of pesticides can either induce or inhibit certain endogenous antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) activities and alter glutathione (GSH) concentrations in rats. The changes in the activities of the antioxidant enzymes can increase the accumulation of reactive oxygen species in tissues and lead to lipid peroxidation. The precursors of oxidative stress are known to eventually cause chronic health in humans occupationally exposed to pesticides. Plant foods contain naturally occurring secondary metabolites such as flavonoids, carotenoids and glucosinolates that have significant beneficial biological effects. Certain flavonoids have been shown to possess antioxidant and free radical scavenging properties, and have been reported to reduce oxidative stress in cells in culture. In this project we will determine the protective effects of food flavonoids in pesticide exposed animal models. Several cell culture studies have shown that flavonoids have both free radical protective properties as well as possess toxic properties at higher concentrations. We have initiated studies in animals orally exposed to a mixture of widely used pesticides and to a combination of pesticides and flavonoids. The pesticide mixture will be fed orally dissolved in oil: acephate, endosulfan and thiram will be fed at 12% of LD50/kg body weight; flavonoids rutin, quercetin and catechin will be added to the diet at 1.0 and 0.1 mM/kg diet. After two weeks the rats will be terminated and endogenous antioxidants will be determined in the blood, liver and small intestines. This study is in progress and could either confirm or refute the observations reported by cell culture studies.

Impacts
We anticipate that the results of this animal study will identify the beneficial effects of flavonoids and also their toxic effects if any, in vivo. The consumption of flavonoids has increased because of their much vaunted protective properties. Therefore, the data will also provide the in vivo biosafety of these phytochemicals.

Publications

  • Panemangalore M and Bebe FN (2005). Interaction between pesticides and essential metal copper increases the accumulation of copper in the kidneys of rats. Biol Trace Elem Res 108: 169-184.
  • Bebe FN and Panemangalore M (2005). Pesticides and essential minerals modify endogenous antioxidants and cytochrome P 450 in tissues of rats. J Environ Sci Hlth (Part B) 40:764-784.


Progress 01/01/04 to 12/31/04

Outputs
Studies in rats have shown that dermal or whole body exposure to low levels of a mixture of pesticides or individual pesticides can either induce or inhibit certain endogenous antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) activities and alter glutathione (GSH) concentrations. The changes in the activities of the antioxidant enzymes can increase the accumulation of reactive oxygen species in tissues and lead to lipid peroxidation. The precursors of oxidative stress can eventually cause chronic diseases in exposed humans. Plant foods contain naturally occurring secondary metabolites such as flavonoids, carotenoids and glucosinolates that have significant beneficial biological effects. Certain flavonoids have been shown to possess antioxidant and free radical scavenging properties and have been reported to reduce oxidative stress in cells in culture. We are determing the protective effects of food flavonoids and are also assessing the concentrations at which these compounds could become toxic, that is their biosafety. We have initiated cell culture studies using Hep G2 cells and experiments are in progress to ascertain their ability to protect cells from oxidative damage induced by pesticides. Some preliminary experiments to mainly set the parameters for further experiments have been completed. We have also conducted duration of exposure and dose response studies. We will test flavonoids quercetin, kaempherol and rutin individually and together at different dosages. These tests are in the process of being repeated for confirmation before we proceed with new experiments.

Impacts
We anticipate that the results will help not only the identify the beneficial effects of flavonoids but also their toxic effects if any. The consumption of flavonoids has increased because of their much vaunted protective properties. Therefore the data from this project will assess the biosafety of these phytochemicals

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
Previous experimental studies in animal models (rats) have demonstrated that dermal as well as whole body exposure to low levels of a mixture of pesticides or individual pesticides can either induce or inhibit certain endogenous antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) activities and alter glutathione (GSH) concentrations. These changes in the activities of the antioxidant enzymes can increase the accumulation of reactive oxygen species in tissues and lead to lipid peroxidation - the precursors of oxidative stress that can eventually cause chronic diseases in exposed humans. Plant foods contain naturally occurring secondary metabolites such as flavonoids, carotenoids and glucosinolates that have significant and perhaps biological effects. Flavonoids and carotenoids have been shown to possess antioxidant and free radical scavenging properties. Certain flavonoids are reported to decrease oxidative stress cells in culture. Certain phytochemicals could protect cells from the oxidative stress induced by pesticides and some could also have detrimental effects at high concentrations. We have initiated cell culture studies using Hep G2 cells to assess the both the antioxidant and toxic effects of phytochemicals and to assess their ability to protect cells from oxidative damage induced by pesticides. Some preliminary experiments to mainly set the parameters for further experiments have been completed. We have also conducted duration of exposure and dose response studies.. We will test flavonoids quercetin, kaempherol and rutin individually and together at different dosages. The data are insufficient to report herein.

Impacts
The consumption of flavonoids has increased because of their much vaunted protective properties, but they have not been tested for their toxic effects. We expect to identify the protective effects of flavonoids on the detrimental effects of pesticide exposure and also assess their possible toxicity at higher doses.

Publications

  • No publications reported this period