Jerry H. Exon

A Review of the Effects and Mechanisms of Polyphenolics in Cancer

This paper is a comprehensive review of the effects of bioactive polyphenolic compounds commonly found in many fruits and vegetables on cancer. These include the pheniolic acids, anthocyanins, catechins, stilbenes and several other flavonoids. We have attempted to compile information from most of the major studies in this area into one source. The review encompasses the occurrence and bioavailability of the polyphenolics, the in vitro and in vivo evidence for their effects on cancer, both positive and negative, and the various mechanisms by which the chemicals may exert their effects. Although most of the work done to date indicates a chemopreventative activity of these compounds, there are some studies that show cancer-inducing or no effects. There are several common mechanisms by which these chemicals exert their effects that could be conducive to additive, synergistic, or antagonistic interactions. These include effects on cellular differentiation, proliferation, and apoptosis, effects on proteins and enzymes that are involved in these processes at a molecular level, and other various effects through altered immune function and chemical metabolism.

Effect of Lead, Polychlorinated Biphenyls, and Cyclophosphamide on Rat Natural Killer Cells, Interleukin 2, and Antibody Synthesis

Interleukin 2 (IL2) activity, natural killer cell (NKC) cytotoxicity, and serum antibody (Ab) levels were assessed in rats exposed to 10 or 1000 ppm lead (Pb) as lead acetate in the drinking water or 50 or 500 ppm polychlorinated biphenyls (PCB) as Aroclor 1254 in the feed for 10 weeks or injected one time with 75 mg/kg cyclophosphamide (CY). Assays for IL2 activity and NKC cytotoxicity were also performed following in vitro exposure of rat splenocytes to Pb (0.4 or 40 micrograms/ml) or PCB (0.4 or 20.0 micrograms/ml) for 24 hr in vitro. NKC cytotoxicity and IL2 activity were significantly suppressed following in vitro exposure to either Pb or PCB. Chronic exposure to PCB, but not Pb, significantly reduced NKC cytolytic activity and significantly elevated Con A-stimulated IL2 activity. Ab synthesis was significantly suppressed in groups of rats chronically exposed to Pb or PCB. CY-injected rats had significantly reduced IL2 activity, NKC cytotoxicity, and Ab levels. High background levels of IL2, presumably induced by KLH injections shortly before termination, were significantly suppressed in PCB-, Pb-, and CY-treated rats. This suppression of IL2 activity was completely reversed by in vitro stimulation with Con A in Pb- or PCB-, but not CY-, treated groups. These results indicate that PCB, Pb, and CY alter IL2 synthesis and adversely affect NKC cytotoxicity and Ab synthesis following in vivo or in vitro exposure. The effects of PCB on NKC cytotoxicity may partially explain the tumor-promoting effect of this chemical via compromising immunosurveillance.

A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity

Many drugs and other chemicals can alter cell-mediated immunity (CMI), a response that often correlates with delayed-type hypersensitivity (DTH). Several DTH assays were evaluated to determine a method best suited for assessing chemically induced modulation of CMI in rats. The effects of various antigens, adjuvants, doses, routes, and immunosuppressants were investigated. The DTH method which produced optimum results in rats uses a footpad swelling reaction elicited by specific preparations of bovine serum albumin (BSA) and Freunds complete adjuvant (FCA). The rats were sensitized with 100 micrograms BSA in FCA injected subcutaneously at the base of the tail, and were challenged 7 days later with 75 microliter of 2% heat-aggregated BSA suspension injected into the left rear footpad. Footpad swelling was measured with pressure calipers 24 h later and compared to the contralateral footpad which was sham-injected with 75 microliters of physiological saline. Antigen-injected footpads were nearly double the thickness (7-8 mm) of the control footpads (3-4 mm), and variation between animals was small (CV = 5%). Dexamethasone and cyclophosphamide significantly suppressed the DTH reaction. Histopathological examination of the DTH reaction sites revealed a mononuclear cell infiltrate which is characteristic of type IV hypersensitivity. In addition to being highly quantitative and sensitive, this method provides a simple and reproducible assessment of CMI responses in the rat.

Subchronic 90 day toxicity of dichloroacetic and trichloroacetic acid in rats

Male Sprague-Dawley rats were treated with either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in the drinking water at levels of 0, 50, 500 and 5000 ppm for a period of 90 days to determine the toxicities associated with subchronic exposure. All animals were sacrificed and examined for gross and histopathologic lesions, serochemical changes, immune dysfunction, hepatic peroxisomal and mixed function oxidase enzyme induction and organ-body weight changes. Animals treated with DCA had decreased body weight gains (500 and 5000 ppm) and decreased total serum protein (all doses). Rats given either TCA (5000 ppm) or DCA (500 or 5000 ppm) had increased liver and kidney organ to body weight ratios. Rats offered DCA had significantly elevated alkaline phosphatase (500 and 5000 ppm) and alanine-amino transferase (5000 ppm). No consistent immunotoxicity was observed in animals exposed to either compound. Rats treated with 5000 ppm TCA or DCA had significantly increased hepatic peroxisomal beta-oxidation activity. These data, along with histopathologic changes, suggest that TCA and DCA produce substantial systemic organ toxicity to the liver and kidney during a 90-day subchronic exposure, although only at doses greater than those expected to occur in the environment.

Dietary Curcumin Enhances Antibody Response in Rats

The effects of dietary curcumin on three major types of immune function were examined in rats. Antibody (IgG) production, delayed-type hypersensitivity and natural killer cell activity were evaluated after 5 weeks of dietary exposure to 1, 20 or 40 mg/kg curcumin. The highest dose of curcumin significantly enhanced IgG levels. Rats receiving lower dietary concentrations (1 or 20 mg/kg) of curcumin were not different in IgG production from rats receiving no curcumin in their diet. Neither delayed-type hypersensitivity nor natural killer cell activity was different from control values at any dietary concentration of curcumin. In vitro incubation of YAC-1 and EL4 tumor cells and normal splenocytes in varying concentrations of curcumin for varying times revealed differences between cell types in curcumins effects on cell proliferation and viability. No cytotoxic effect was seen in EL4 cells at 125 micrograms/ml curcumin at 4, 24 and 48 hrs incubations, however, cell proliferation was reduced by almost 50% at 24 hrs. YAC-1 cell viability and cell numbers were diminished at longer incubations. A lower curcumin concentration (1.25 micrograms/ml) enhanced cell growth in the YAC-1 cells at 24 and 48 hr. This enhancement was not seen in spleen or EL4 cells.

Dietary Quercetin, Immune Functions and Colonic Carcinogenesis in Rats

Rats fed 100 mg/kg quercetin (QUE) daily for 7 weeks had significantly enhanced natural killer cell activity compared to their vehicle (VEH)-fed control. In contrast, rats fed 100 mg/kg QUE and treated with the colon carcinogen, azoxymethane had significantly reduced natural killer cell activity compared to their VEH-fed azoxymethane-treated control. There was no significant difference in natural killer cell activity between the two control groups. Antibody production and delayed-type hypersensitivity were not altered by QUE feeding in any treatment group. In vitro exposure of splenic natural killer cells to 1mM QUE significantly decreased natural killer cell cytotoxicity. Lower QUE concentrations produced a non-significant reduction in natural killer cell activity that was restored to control values at 1 x 10(-13)M QUE. The distribution, multiplicity and total number of colonic preneoplastic lesions, aberrant crypt foci, was not significantly different in the QUE-fed azoxymethane-treated rats when compared to azoxymethane-treated vehicle-fed rats at the conclusion of 7 week feeding period. We found no correlation between immune function and development of preneoplastic colon lesions in this study.

Effects of subchronic exposure of rats to 2-methoxyethanol or 2-butoxyethanol: Thymic atrophy and immunotoxicity☆

Male Sprague-Dawley rats were exposed to either 2000 or 6000 ppm of 2-methoxyethanol (ME) or 2-butoxyethanol (BE) and females were exposed to either 1600 or 4800 ppm of these compounds in the drinking water for 21 days. Body weights were decreased in male rats exposed to the high doses of both chemicals, while body weights of females exposed to either dose of BE were decreased. Male and female rats exposed to either concentration of ME had a dose-related reduction in thymus weights. Testis weight was significantly lower in male rats exposed to the high dose of ME. Dose-related increases in natural killer (NK) cell cytotoxic activities and decreases in specific antibody production were observed in all rats treated with ME. Rats exposed to the low dose of BE also had enhanced NK cell activity. Splenocyte production of interferon-gamma was decreased in male rats exposed to either dose of ME and in females treated with the high dose of ME. Spleen cell numbers were reduced in males exposed to the high dose of ME and females given either dose of ME. It appears that the immune system is a sensitive target of ME but not BE. The effects of ME on immune function differ depending on the immune parameter assessed. Enhanced NK cell activity may partially explain the observations of others that certain glycol ethers have antitumor effects in vivo.

Alteration of natural killer cell-mediated cytotoxicity in rats treated with selenium, diethylnitrosamine and ethylnitrosourea

Weanling, female Sprague--Dawley rats were divided into 14 separate groups. Three of these groups were administered 0.5, 2.0 or 5.0 ppm selenium (Se) in the drinking water for 10 weeks. Three groups received intraperitoneal injections of 1, 5 or 10 mg/kg diethylnitrosamine (DEN) twice weekly for 10 weeks. The remaining animals received 0.150% or 0.316% ethylurea (EU) in the feed and 1 or 10 ppm nitrite as sodium nitrite in the drinking water either alone or in combination. Separate groups of rats treated with cyclophosphamide (CY) were included as positive immuno-suppressed controls. Following the 10-week chemical exposure period, splenic natural killer (NK) cell-mediated cytotoxicity was assessed by a 4-h chromium release assay using YAC-1 tumor cells as targets. The NK cell cytotoxic response was enhanced in both the low and medium dose selenium-exposed groups. In contrast, rats exposed to 0.316% EU + 10 ppm NO2 had significantly depressed NK cell activity. CY treatment also resulted in a significant reduction of splenic NK cell cytotoxicity.

Effects of chlorinated phenols on immunity in rats

Female Sprague - Dawley rats were exposed to 0, 5, 50 or 500 ppm 2-chlorophenol (2-CP) or pentachlorophenol (PCP) from weaning to 3 weeks postparturition after breeding at 90 days of age. Progeny were weaned at 3 weeks of age and continued on chlorophenol treatment for 10 weeks at which time major immune functions were tested. Humoral immunity was measured by an indirect ELISA, cell-mediated immunity was monitored by delayed-type hypersensitivity (DTH) to oxazolone, and macrophage function was tested by phagocytosis of sheep red blood cells. Rats treated with cyclophosphamide were included as a positive immunosuppressed control. PCP-treated rats had significantly decreased antibody titers and DTH response and increased induced peritoneal macrophage numbers which displayed hyperphagocytic activity. Immune responses in rats treated with 2-CP were not significantly different from controls. The data indicate that (1) the immune system may be a sensitive target for PCP toxicity but not for 2-CP, (2) closely related chlorophenolic chemical isomers may exert different toxic effects on the immune system, and (3) PCP can exert depressive effects on some major immune parameters while enhancing others.

MULTIPLE IMMUNE FUNCTIONS IN RATS FED ECHINACEA EXTRACTS

This study evaluated acquired-immune functions representing the three major branches of the immune system in male rats fed a commercially available echinacea product. An additional comparison of effects on antibody formation in male and female rats was done using the commercial echinacea product and two echinacea tinctures marketed by local herbalists. In initial testing, we found no evidence of altered natural killer cell activity, T cell-mediated delayed-type hypersensitivity, or specific antibody formation in male rats given either a 225 mg/kg or 50 mg/kg of the commercial echinacea for 6 weeks. Antibody formation was significantly suppressed in female but not male rats given 250 mg/kg for 2 weeks of the commercial echinacea. The local products tested had no effect on antibody formation. We concluded that our study provided no supporting evidence for immunostimulatory activity by the echinacea preparations we examined and, in fact, may be immunosuppressive under some conditions.