Missed opportunities for prevention: smoking cessation counseling and the competing demands of practice

Activating mutations in and expression of the Ha-ras gene were examined in benign and malignant female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a diet high in polyunsaturated fat. Ha-ras mutations were detected in codons 12 and 13 by selective polymerase chain reaction amplification of mutated sequences and nucleotide sequencing. The percentage of Ha-ras mutations in carcinomas from PhIP-treated rats was significantly higher in rats on a low-fat diet than in rats on a high-fat diet (82% (nine of 11) vs 26% (seven of 27), respectively, P < 0.01). In addition, whereas 56% of the carcinomas with Ha-ras mutations from rats on a low-fat diet carried double Ha-ras mutations, none of the carcinomas from rats on a high-fat diet had double mutations. Ha-ras mutations were also detected in benign tumors (largely adenomas) induced by PhIP in rats on different diets; two of eight and three of four benign tumors examined from rats on low-fat and high-fat diets, respectively, had Ha-ras mutations, suggesting that activating Ha-ras mutations alone are not sufficient for PhIP-induced tumors to become malignant. No differences were observed in the level of Ha-ras mRNA expression in the different groups. In our animal model, a high-fat diet increased the incidence and percentage of malignant PhIP-induced mammary gland tumors yet decreased the percentage of carcinomas showing Ha-ras mutations. Thus, the complement of genetic alterations associated with PhIP-induced mammary gland carcinogenesis is probably altered by the level of dietary fat.     

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.     

Inhibitory effects of diallyl disulfide or aspirin on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary carcinogenesis in rats

Modifying effects of diallyl disulfide (DAD), aspirin or DL-alpha-difluoromethylornithine (DFMO) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in SD rats were investigated. A total of 166 female rats, 6 weeks old, were divided into 8 groups. They were fed a high fat diet throughout the experiment. Starting at 7 weeks of age, groups 1-4 were given PhIP (85 mg/kg body weight in corn oil) by gavage 8 times in 10 days, and groups 5-8 were given corn oil alone. For the beginning 4 weeks, groups 2 and 5 were given DAD at 200 ppm in diet. Similarly groups 3 and 6, and groups 4 and 7 were given aspirin (400 ppm) and DFMO (400 ppm), respectively. Mammary carcinomas were only recognized in groups 1-4 at the termination (25 weeks after the start of experiment). Multiplicity (mean number/rat) of neoplasms in group 2 (PhIP+DAD, 0.90/rat) and group 3 (PhIP+aspirin, 1.37/rat) was significantly smaller than that in group 1 (PhIP alone, 2.45/ rat) (P < 0.005 and P < 0.05, respectively). These results indicate that dietary intake of DAD or aspirin during the time corresponding to initiation phase has chemopreventive potential on PhIP-induced mammary carcinogenesis in rats.     

Differential effect of triiodothyronine and thyroxine on liposomes containing cholesterol: physiological speculations

Chemopreventive effects of the antioxidants 1-O-hexyl-2,3,5- trimethylhydroquinone (HTHQ), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethylascorbic acid (DAsA), green tea catechins (GTC) and ellagic acid on 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were examined in female F344 rats. Groups of 20-21 6-week-old rats were maintained on a powdered diet containing 0.02% PhIP alone, PhIP together with 0.5% HTHQ, 1% EAsA, 1% DAsA, 1% GTC or 0.1% ellagic acid, these antioxidants alone or basal diet alone without supplement for 52 weeks. The survival rates of PhIP plus antioxidant groups at the end of the experiment were higher than that of the PhIP alone group. Sequential observation of palpable mammary tumors demonstrated only one tumor by week 52 in the PhIP plus HTHQ group, whereas 40% of the rats receiving PhIP alone had tumors by this time point. The final incidence of mammary adenocarcinomas was significantly decreased in the PhIP plus HTHQ group (4.8%, P < 0.01) as compared to the PhIP alone value (40%). Although statistically not significant, incidences of adenocarcinomas in the other antioxidant-treated groups (23.8-28.6%) were also lower than in the PhIP alone group. Furthermore, the incidence of large intestinal tumors in the PhIP plus HTHQ group (0%) showed a tendency to decrease relative to the PhIP alone group (16.7%). These results indicate that antioxidants, particularly HTHQ, exert a potent chemopreventive action against PhIP-induced carcinogenesis.     

Bioactivation of the proximal food mutagen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) to DNA-binding species by human mammary gland enzymes

We have investigated phase II activation of the food-derived mutagen 2-hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding of calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.     

Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro

Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas.     

Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP-dG, PhIP-DNA and DiMeIQx-DNA from the rat

The heterocyclic aromatic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are formed during frying of meat. PhIP and 4,8-DiMeIQx have, after metabolic activation, been shown to form adducts with DNA at the C8 of guanine both in vitro and in vivo. In order to investigate possible urinary biomarkers for estimation of the genotoxic dose of PhIP and 4,8-DiMeIQx, [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA were injected i.p. to rats and the excretion of radioactivity in urine and faeces were measured. For all three [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA 15-20% of the dose were excreted in the urine and 80-85% of the dose were excreted in the faeces. Urinary excretion showed maximum to 24 h (90%) with a rapid decline, 10% to 48 h and 0% to 72 h. Faecal excretion also showed maximum to 24 h (60%) with a slower decline, 30% to 48 h and 10% to 72 h. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-dG, showed that approximately 90% of the radioactivity co-eluted with PhIP-dG, indicating that PhIP-dG is excreted unmetabolized. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-DNA, showed that approximately 85% of the radioactivity co-eluted with PhIP-dG, indicating that PhIP-DNA adducts is mainly excreted as nucleoside adducts. Approximately 5% of the radioactivity excreted in the urine co-eluted with PhIP-G, indicating loss of deoxyribose. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [14C]4,8-DiMeIQx-DNA, showed that approximately 90% of the radioactivity co-eluted with 4,8-DiMeIQx-dG, indicating that 4,8-DiMeIQx-DNA adducts is mainly excreted as nucleoside adducts. Man is able to eliminate compounds of a higher mol. wt in the urine than the rat, the percentage of PhIP-dG and 4,8-DiMeIQx eliminated in the urine of man would therefore be expected to be higher than in the rat. Measurement of urinary nucleoside adducts of PhIP and 4,8-DiMeIQx could therefore provide a basis for the development of a biomonitoring strategy for the genotoxic dose of these food derived HAA.     

Effect of exercise and tepary bean type diet on body composition and fat accretion in obese Zucker rats

OBJECTIVE: The effectiveness of a tepary bean high fat type diet, compared to a purified type high fat diet and exercise, on body composition in fatty Zucker rats was determined. SUBJECTS AND DESIGN: Approximately 6-week-old female fa/fa Zucker rats were divided into four groups of 10 rats each: TE, fed the tepary bean type diet and exercised; TN, fed the tepary bean type diet and not exercised; CE, fed the purified type control diet and exercised; CN, fed the purified type control diet and not exercised. The exercise modality was treadmill running and the experiment lasted 13 weeks. MEASUREMENTS: Body weight, cumulative food intake, body composition, weights of adipose tissues and liver, heart and gastrocnemius muscle. RESULTS: At the end of the 13 week experiment, TE rats weighed 511 +/- 22 g and were significantly lighter than TN, 588 +/- 15 g; CE, 606 +/- 22 g; and CN, 660 +/- 27 g. All are means +/- s.e.m. The carcass of CN rats had 58, 20 and 13% more fat than TE, TN and CE rats, respectively; P < 0.01. Lean body mass was the same for all the groups of rats and ranged from means of 216-228 g. However, TE rats had significantly more fat free dry mass (FFDM) than CN rats; 68 +/- 4 vs 58 +/- 2 (means +/- s.e.m.) and tended to have more FFDM than TN and CE rats. Inguinal fat depots weighed 20-30% less in T than in C rats (diet comparisons) and also 20-30% less in E than in N rats (exercise comparisons). Perirenal/retroperitoneal fat depots weighed 25% less in TN than in CN rats and 38% less in TE than in CE rats. Exercise did not reduce perirenal/retroperitoneal fat depot weights. Parametrial fat depot weights were not influenced by diet or exercise. CONCLUSIONS: In diets which provided 37% of the energy from fat, the incorporation of tepary beans attenuated weight gain, and subcutaneous and visceral fat gain compared to a purified type diet. Exercised rats gained less weight and subcutaneous, but not visceral fat, than non-exercised rats.     

Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N-acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N-hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N-acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.     

Structural vascular changes in hypertension: role of angiotensin II, dietary sodium supplementation, blood pressure, and time

Previous studies have shown that a diet high in polyunsaturated fatty acids increases mammary tumor incidence in adult and pregnant mice and rats and in the female offspring. The present study investigated whether a high-fat diet alters the number of estrogen receptor (ER) binding sites and protein kinase C (PKC) activity in the mammary gland of these animals. In the female offspring, the effects of maternal exposure to a high-fat diet during pregnancy on development of the mammary epithelial tree were studied also. BALB/c mice were kept on a diet containing either 43% (high-fat) or 16% (low-fat) calories from corn oil, which consists mostly of n-6 polyunsaturated fatty acids, for 1 month. In adult female mice, a 6-fold increase in the number of ER binding sites and 2-fold increase in PKC activity were found in the mammary glands of the high-fat mice when compared with the low-fat mice. In pregnant mice, a high-fat diet increased ER binding sites by 61% and PKC activity by 51%. In contrast to adult and pregnant mice, females exposed to a high-fat diet only in utero through their pregnant mother exhibited a significantly reduced number of mammary ER binding sites by age 45 days (78% decrease) and a reduction in PKC activity by ages 30 and 100 days (44 and 20% decrease, respectively). The mammary epithelial tree of the high-fat offspring contained more terminal end buds and was less differentiated than that of the low-fat offspring. These findings show that consumption of a high-fat diet increases ER and PKC in the adult and pregnant mouse mammary gland, perhaps contributing to the fat-induced promotion of mammary tumorigenesis. In contrast, reduced ER and PKC following a high-fat exposure in utero may be associated with increased susceptibility to carcinogenesis, possibly due to an increased number of terminal end buds that are the sites of neoplastic transformation in the mammary gland.     

An entire functional mammary gland may comprise the progeny from a single cell

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore, one stem cell might easily account for the renewal of mammary epithelium over several transplant generations.     

The metabolism and DNA binding of the cooked-food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in precision-cut rat liver slices

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.     

Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk

Previous research showed that treatment with conjugated linoleic acid (CLA) during the period of active mammary gland morphogenesis was sufficient to confer a lasting protection against subsequent mammary tumorigenesis induced by methylnitrosourea. The present study was designed to characterize certain morphological and biochemical changes of the mammary gland that might potentially render it less susceptible to cancer induction. Female Sprague Dawley rats were fed a 1% CLA diet from weaning until about 50 days of age. The mammary gland parameters under investigation included (a) the deposition of neutral lipid, (b) the identification and quantification of CLA and its metabolites, (c) the density of the epithelium, and (d) the proliferative activity of various structural components. Our results showed that CLA treatment did not affect total fat deposition in the mammary tissue nor the extent of epithelial invasion into the surrounding fat pad but was able to cause a 20% reduction in the density of the ductal-lobular tree as determined by digitized image analysis of the whole mounts. This was accompanied by a suppression of bromodeoxyuridine labeling in the terminal end buds and lobuloalveolar buds. The recovery of desaturation and elongation products of CLA in the mammary gland confirmed our prior suggestion that the metabolism of CLA might be critical to risk modulation. The significance of the above findings was investigated in a mammary carcinogenesis bioassay with the use of the dimethylbenz[a]anthracene model. When CLA was started at weaning and continued for 6 months until the end of the experiment, this schedule of supplementation produced essentially the same magnitude of mammary tumor inhibition in the dimethylbenz[a]anthracene model as that produced by 1 month of CLA feeding from weaning. The observation is consistent with the hypothesis that exposure to CLA during the time of mammary gland maturation may modify the developmental potential of a subset of target cells that are normally susceptible to carcinogen-induced transformation.     

Black tea and mammary gland carcinogenesis by 7,12-dimethylbenz[a]anthracene in rats fed control or high fat diets

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor-bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.     

Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland

The involvement of glycoprotein CD36 and fatty-acid-binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C-terminus (amino acid residues 121-131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti-FABP and anti-CD36, Northern-blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C-terminus of FABP with the sequence Val-Thr-Cys, identical to the sequence found in the CD36-binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val-Cys-Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period.     

Transcellular transport of benzoic acid across Caco-2 cells by a pH-dependent and carrier-mediated transport mechanism

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are two members of a family of carcinogenic heterocyclic amines (HAs) found in cooked meats that form DNA adducts after activation to N-acetoxy derivatives. The ability of IQ- and PhIP-DNA adducts to inhibit gene expression was investigated using a human growth hormone (hGH) reporter gene in a pUC12-based mammalian expression vector under the control of either the herpes simplex virus-1 thymidine kinase promoter or the human immunodeficiency virus-1 long terminal repeat. The plasmids were treated in vitro with 0, 5, 10, or 40 microM N-hydroxy-IQ or N-hydroxy-PhIP in the presence of a 10-fold molar excess of acetic anhydride to generate the N-acetoxy derivatives in situ. The adduct levels in the plasmids were quantitated by the 32P-postlabeling method. The adducted (and control) plasmids were each transfected into repair-deficient or -proficient Chinese hamster ovary cells, and expression of hGH was measured by immunoassay of growth hormone secreted into the cell medium. The results showed that IQ- and PhIP-DNA adducts inhibited gene expression in both plasmids and that the degree of inhibition of hGH production was proportional to the levels of IQ- and PhIP-DNA adducts. The degree of inhibition, however, was independent of the promoter, despite the differences in the strengths of the two promoters to drive hGH production. Repair capacity influenced the extent of inhibition of gene expression by HA adducts since, in general, fewer adducts were needed to inhibit reporter gene expression in repair-deficient cells than in repair-proficient cells. In both cell lines, DNA adducts of PhIP appeared to be more potent in inhibiting hGH expression than adducts of IQ. Whether alteration of gene expression by HA adducts plays a role in the carcinogenicity of these compounds deserves further study.     

Antibodies to the food mutagens, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline: useful for immunoassay and immunoaffinity chromatography of biological samples

Monoclonal mouse IgG1 and IgG3 antibodies were developed to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,4-f] quinoxaline (4,8-DiMeIQx) in order to make specific and sensitive detection and purification systems suitable for biological samples. The antibodies were developed with the strategy that cross-reaction with analogues modified in the N2-position was desirable. Competitive enzyme-linked immunosorbent assays (ELISA) with 50% inhibition by 0.4-6 pmol food mutagen were developed. The epitopes recognized by the antibodies have been characterized by ELISA using 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx, and other food mutagens. One of the anti-PhIP antibodies only recognizes PhIP and those PhIP-analogues which have minor modifications in the N2-amino group, whereas the other, 7B7-1, is less stringent and also recognizes several other modified metabolites, including bulky adducts at the N2-amino group e.g. the major guanine and deoxyguanosine adducts isolated from PhIP-modified DNA. The antibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (4-MeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two of these antibodies only bind analogues with minor modifications in the free amino group, whereas analogues with major modifications in this position, including a deoxyguanosine adduct, react with the third antibody. Urine samples and faecal extracts from 3H-PhIP or 2-14C-DiMeIQx dosed rats were analysed by these ELISA assays, and high correlations between radioactivity and response in the ELISA assays were observed. Urine samples and faecal extracts from 3H-PhIP-dosed rats were purified on an affinity column containing the less stringent anti-PhIP antibody, 7B7-1. The affinity column was found by high performance liquid chromatography (HPLC) analysis to concentrate exclusively labelled material. This affinity column also bound PhIP-related materials from dilute samples of acid hydrolysed PhIP-DNA with high efficiency. Only approximately 40% of the 4,8-DiMeIQx related materials found in dilute acid hydrolysed samples of 4,8-DiMeIQx-DNA was bound by an affinity column containing the less stringent anti-4,8-DiMeIQx antibody, 2C5-1. We conclude that our anti-PhIP and anti-DiMeIQx antibodies can be used to determine the presence of these food mutagens and some of their activated or conjugated metabolites in complex biological samples.     

Antagonism of l-stepholidine against bromocriptine-inhibition on prolactin level in lactational rats

AIM: To study the antagonism of l-stepholidine (SPD) against bromocriptine (Bro)-inhibition on prolactin (PRL) level. METHODS: Bro (0.5 mg.kg-1.d-1, s.c.) reduced the PRL and caused a dysplasia of mammary gland in lactational rats. The weight growing of newborn rats was retarded. The PRL of the lactational rats was assessed by immunoradiometric assay (IRMA); the weight of newborn rats and development of mammary glands in lactational rats were also examined. Antagonism of SPD was evaluated. RESULTS: SPD (30 & 100 mg.kg-1.d-1, i.p.) obviously antagonized the Bro that induced lowering the PRL level in lactational rats, the PRL was 11 +/- 4 & 23 +/- 6 micrograms.L-1 (NS 7 +/- 2) respectively on d 15 of postpartum and the development of mammary gland in lactational rats was normal. The newborn rats grew rapidly in 11-15 d. CONCLUSION: SPD possessed an antagonism with Bro inhibition on D2 receptors located in the pituitary gland, and was an antagonist of dopamine D2 receptors.