Edward G. Miller

Differential Inhibition of Human Cancer Cell Proliferation by Citrus Limonoids

Limonoids have been shown to inhibit the growth of estrogen receptor-negative and -positive human breast cancer cells in culture. The primary objective of this study was to test the antiproliferative activity of limonoids (obacunone 17β-D-glucopyranoside, nomilinic acid 17β-D-glucopyranoside, limonin, nomilin, and a limonoid glucoside mixture), found in high concentrations in mandarin (Citrus reticulata Blanco), against a series of human cancer cell lines. The human cancer cell lines included leukemia (HL-60), ovary (SKOV-3), cervix (HeLa), stomach (NCI-SNU-1), liver (Hep G2), and breast (MCF-7). The growth-inhibitory effects of the four limonoids and the limonoid glucoside mixture against MCF-7 cells were significant, and the antiproliferative activity of the different citrus limonoids was also dose and time dependent. No significant effects were observed on growth of the other cancer cell lines treated with the four individual limonoids at 100 μg/ml. At 100 μg/ml, the limonoid glucoside mixture demonstrated a partial inhibitory effect on SKOV-3 cancer cells. With use of flow cytometry, it was found that all the limonoid samples could induce apoptosis in MCF-7 cells at relatively high concentrations (100 μg/ml). Considering the high concentration needed to induce apoptosis, it is unlikely that this is the primary mechanism of action for the cytotoxic effects seen with limonoids in this study. Further work is needed in this area to establish the mechanism of action of citrus limonoids on human breast cancer cells.

Stimulation of nuclear poly (adenosine diphosphate-ribose) polymerase activity from HeLa cells by endonucleases.

Isolated nuclei from HeLa cells can incorporate labeled ADP-ribose from NAD into an acid-precipitable product, poly(ADP-ribose). This reaction is stimulated by 4-6-fold by the addition of deoxyribonuclease I to the complete reaction mixture. If the nuclei are treated first with deoxyribonuclease I, no effect is seen; the stimulation is only apparent when the two enzymes deoxyribonuclease I and poly(ADP-ribose) polymerase, are operating at the same time. After making several minor modifications in the assay mixture, it was found that another endonuclease, micrococcal nuclease, can also stimulate the poly(ADP-ribose) polymerase activity of HeLa nuclei. A comparison of the two stimulatory effects indicated that the two endonucleases activated to the poly(ADP-ribose) polymerase activity of HeLa nuclei in the same way. Overall this evidence suggests that poly(ADP-ribose) polymerase may have a functional role in the process of DNA repair.

Inhibition of hamster buccal pouch carcinogenesis by limonin 17-β-D-glucopyranoside

Limonin 17-beta-D-glucopyranoside, nomilin 17-beta-D-glucopyranoside, and nomilinic acid 17-beta-D-glucopyranoside, three limonoid glucosides isolated from oranges, were tested for cancer chemopreventive activity. Eighty female Syrian hamsters were divided into four equal groups. The left buccal pouches of the animals in each group were pretreated topically with two applications of water (Group I) or a 3.5% solution of limonin 17-beta-D-glucopyranoside (Group II), nomilin 17-beta-D-glucopyranoside (Group III), or nomilinic acid 17-beta-D-glucopyranoside (Group IV). After this initial treatment, the left buccal pouches of 16 hamsters from each group were painted five times per week. Two or three times per week the pouches were treated with a 0.5% solution of the carcinogen 7,12-di-methylbenz[a]anthracene (DMBA) dissolved in mineral oil. On alternate days, the pouches were treated with water (Group I) or a 3.5% solution of limonin 17-beta-D-glucopyranoside (Group II), nomilin 17-beta-D-glucopyranoside, or nomilinic acid 17-beta-D-glucopyranoside. The 16 remaining animals were used as controls. These hamsters were treated five times per week, one day with mineral oil and the next with either water (Group I) or one of the 3.5% solutions of the limonoid glucosides (Groups II-IV). After 15 weeks (71 applications), the hamsters were killed. Multiple tumors were common in the animals treated with DMBA; however, the animals treated with limonin 17-beta-D-glucopyranoside exhibited a 55% decrease in average tumor burden. Further comparisons between Groups I and II showed that this reduction in tumor burden was mainly due to a decrease in tumor mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Oral Carcinogenesis by Citrus Flavonoids

Six citrus flavonoids were tested for antineoplastic activity. The hamster cheek pouch model was utilized, and the solutions of the flavonoids (2.0–2.5%) and the solution of the carcinogen, 7,12-dimethylbenz[a]anthracene (0.5%), were applied topically to the pouches. The pouches of the positive controls were treated with the solvent used to dissolve the flavonoids and the solution of the carcinogen. The data show that 4 flavonoids (hesperetin, neohesperidin, tangeretin, and nobiletin) were inactive. The results with naringin and naringenin show that both of these flavonoids significantly lowered tumor number [5.00 (control group), 2.53 (naringin group), and 3.25 (naringenin group)]. Naringin also significantly reduced tumor burden [269 mmm3(control group) and 77.1 mmm3(naringin group)]. The data suggest that naringin and naringenin, 2 flavonoids found in high concentrations in grapefruit, may be able to inhibit the development of cancer.

Effect of deoxyribonuclease I on the number and length of chains of poly(ADP-ribose) synthesized, in vitro

Summary The effect of DNAase I on the synthesis of poly(ADP-ribose) by HeLa nuclei was studied. Experiments with the isolated products showed that the addition of DNAase I to the complete reaction mixture increased the chain length of the isolated product, poly(ADP-ribose), by only 10–20%. This small increase cannot account for the 400–600% increase in poly(ADP-ribose) polymerase activity that is seen when DNAase I is added to the assay media. So these results indicate that the main effect of DNAase I on the synthesis of poly(ADP-ribose) is through the initiation of new chains of poly(ADP-ribose). This evidence supports the idea that poly(ADP-ribose) polymerase may have a role in DNA repair.

Kahweol and cafestol: Inhibitors of hamster buccal pouch carcinogenesis

Kahweol and cafestol, two compounds extracted from green coffee beans, were tested for cancer chemopreventive activity. For the experiment, 60 hamsters were divided into three equal groups and placed on one of three diets. The animals in Group I received a normal diet, whereas the animals in Groups II and III received the same diet supplemented with a 50:50 mixture of kahweol and cafestol. The content of the kahweol and cafestol mixture in these two diets was 0.2 g/kg of food (Group II) and 2.0 g/kg of food (Group III). After the hamsters adjusted to their respective diets, 16 hamsters from each group were selected. The left buccal pouches of these animals were painted three times weekly with a 0.5% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in mineral oil. The 12 remaining hamsters were used as controls. The left buccal pouches of these animals were painted three times weekly with mineral oil. After 13 weeks (39 applications) the hamsters were killed. Multiple tumors were common in animals treated with DMBA; however, the animals receiving kahweol and cafestol in the diet (2 g/kg of food) exhibited a 35% reduction in tumor burden. Further comparisons between Groups I and III showed that this reduction in tumor burden was due to a decrease in tumor number. The results for Group II were inconclusive. Some reduction in tumor number was found, but this was offset by an increase in the size of the tumors.

Inhibition of hamster buccal pouch carcinogenesis by green coffee beans

Forty female Syrian Golden hamsters were divided into two equal groups and placed on one of two diets. The hamsters in Group I received a normal chow, and the animals in Group II were given the same chow supplemented with powdered green coffee beans (20%). After the hamsters adjusted to the diets, 16 animals from each group were selected. The right buccal pouch of each of these hamsters was painted three times weekly with a 0.5% solution of 7, 12-dimethylbenz(a)anthracene (DMBA) in heavy mineral oil. The eight remaining animals, four in each group, served as controls and were treated three times weekly with heavy mineral oil. After a total of 50 treatments (16 1/2 weeks), the hamsters were killed. The animals receiving green coffee beans exhibited a significant delay in the development of DMBA-induced carcinogenesis of the buccal pouch mucosa, as determined by both visual and histologic examination.

In vivo studies on the carcinogenic potential of an orthodontic bonding resin

Data from another laboratory have indicated that the individual components of an orthodontic bonding resin might contain a carcinogen. Since that report, the formulation of the product was changed. The purpose of this study was to determine whether the new product is safe. Three groups of rats were used for the experiment. One group served as a control, while the other groups ingested the sealant resin or sealant catalyst. The materials were suspended in an alcohol-aqueous mixture and the solutions were given to the animals as their only source of fluid. The exposure was for 1 year. After this period of time, all the rats were given tap water and observed until day 600. The animals were autopsied at time of death or at the end of the experiment. During the treatment, there were significant differences (p less than 0.01) in water intake among the three groups. The average intake per day for the animals in the control group, the resin group, and the catalyst group was 50.2 cc, 37.8 cc, and 42.2 cc, respectively. Several animals died during the experiment, but there was no significant differences in the life expectancy of the animals in the three groups. The autopsies uncovered one malignant neoplasm, an undifferentiated sarcoma, in a rat from the control group and four benign tumors in rats from the three groups. All of these results indicate that the new formulation of the orthodontic bonding resin is not carcinogenic when ingested at a dose level of 50 ppm.

Prevalence of mercury hypersensitivity in dental students

F or approximately 150 years, mercury in amalgam has been used as a dental restorative material. Throughout this time the potential toxicity of this metal to patients’” and dental professionals4-9 has been studied and debated. In these reports and many others, problems with mercury contamination and toxicity were uncovered. Although the extent of the problems may be controversial, the overall impact has led to increased emphasis on mercury hygiene in dental schools and in private practice.‘0,1’ Most studies on mercury contamination have concentrated on patients and dentists, with considerably less attention to dental students. This is unfortunate because students, in a sense, serve as a bridge between the two major groups, practicing dentists and their patients. Certainly, problems with mercruy contamination in first-year dental students should be similar to problems in patients. Conversely, problems with mercury contamination or toxicity in fourth-year dental students should reflect problems in practicing dentists. Some attempts have been made to study this unique group. In most of these experiments, however, the number of students screened for different forms of mercury contamination or toxicity has been small, with numbers ranging from 8 to 29 students.‘2-‘6 For this reason, there is little agreement between the results of these pilot studies. In a study by White and Brandt,” however, 298 dental students were screened for allergic responses to mercury. Through the use of epicutaneous patch-test procedures, they showed that the rate of hypersensitivity to mercuric chloride increased as students progressed through the dental curriculum. This observation linking hypersensitivity to mercury to the preparation of alloy restorations suggested that mercury as an allergen was an additional occupational hazard for the dental profession. With increased emphasis on mercury hygiene and the development of improved techniques for preparing amalgam, a second study of this phenomenom was

3-Methoxybenzamide, A Possible Initiator for DMBA-Induced Carcinogenesis

During the last few years this laboratory has formulated a hypothesis (Fig. 1) linking nicotinamide, NAD, and poly(ADP-ribose) polymerase to carcinogenesis. The factors that have led to the development of this hypothesis are as follows: (i) The fact that poly(ADP-ribose) polymerase utilizes NAD as a substrate to form poly(ADP-ribose) (1). (ii) The ability of this nuclear enzyme to respond to damage in DNA (2–4). (iii) Evidence (5, 6) indicating that poly(ADP-ribose) polymerase has a role in DNA repair, (iv) The fact that carcinogens damage DNA and the possibility that this damage is the driving force in carcinogenesis (7, 8).