[Photographs of deceased newborn infants as documentation and as aid in mourning process for parents]. / Foto's van overleden pasgeboren kinderen voor de ouders als documentatie en als hulpmiddel bij de rouw.

The death of a newborn infant is an extremely emotional event for relatives. Many hospitals provide the parents with support, and in some cases a mourning protocol is available. Some hospitals offer parents photographs of infants which have died around birth. The photographs are often taken by a nurse or doctor on the maternity or paediatric ward. It is advisable to draw up a mourning protocol which allows professional studio photographs to be taken. As a photograph is often the only concrete memento that parents have of their baby, it is important for it to be of good quality. The parents decide what they would prefer, but it is important to point out to them that their wishes and needs immediately following the death may differ to those in the longer term.

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice.

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P < 0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6+/-0.6 vs 4.6+/-0.7, respectively, relative adduct labeling (RAL) x 10(8), mean+/-S.E.M., n = 3-5 mice). In the mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9+/-1.1 and 22.5+/-6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 10(9)). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1+/-9.5 and 58.0+/-21.7, respectively, but accordingly only 3.8+/-0.7 and 5.4+/-0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.

Intestinal tumorigenesis in multiple intestinal neoplasia mice induced by the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: perinatal susceptibility, regional variation, and correlation with DNA adducts.

In our previous experiments, multiple injections with the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were used to induce intestinal tumors in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. To define the period of highest susceptibility to PhIP perinatally, we first determined the effect of a single s.c. injection. Ten or 50 mg/kg PhIP increased the number and diameter of small intestinal tumors dose-dependently in 3-day-old Min/+ mice. In the colon, only 50 mg/kg PhIP increased the incidence and number of tumors. The number of dysplastic aberrant crypt foci decreased from weeks 7 to 11. In the same period, an increase in the number of tumors was seen, indicating that over time the dysplastic aberrant crypt foci develop into tumors. Min/+ mice were then exposed in utero through their dams being given one s.c. injection of 50 mg/kg PhIP 3 days before giving birth or were exposed directly to the same dose on day 3, 12, or 36 after birth. Remarkably, the most susceptible period for tumorigenesis in the small intestine was between days 3-12 after birth, whereas in the colon it was from day 3 before to day 3 after birth. Furthermore, we examined whether the formation of DNA adducts determined after 24 h could explain the observed time-dependent and regional susceptibility to PhIP. A higher level of PhIP-DNA adducts was found after exposure on day 12 after birth, compared with day 36 after birth, in all parts of the small intestine but not in the colon, which was in close accordance with the numbers of tumors present. The levels of PhIP-DNA adducts along the intestines were highest in the middle and distal parts of the small intestine, where tumor numbers were also the highest. In conclusion, Min/+ mice are most susceptible to intestinal tumor induction by PhIP from day 3 before to day 12 after birth, and this susceptibility could at least partly be explained by the formation of PhIP-DNA adducts.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) modulates lactogenic hormone-mediated differentiation and gene expression in HC11 mouse mammary epithelial cells.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen in cooked meat. Using the HC11 mouse mammary epithelial cell line, a well-characterized model for hormone-mediated differentiation, we examined whether PhIP altered the expression of genes regulated by lactogenic hormones dexamethasone, insulin, and prolactin (DIP). When HC11-Lux cells (stably transfected with a beta-casein promoter luciferase construct) were cultured in DIP-containing medium, PhIP (100 microM) enhanced luciferase activity 11-fold over that observed in DIP medium alone. The effect of PhIP on augmenting luciferase activity was observed only when lactogenic hormones were included in the medium. Expression of the endogenous beta-casein gene was also higher in HC11 cells treated with PhIP in hormone-enriched medium. With the increased expression of beta-casein gene, the level of phospho-signal transducer and activator of transcription 5A (phospho-STAT5A), the transcription factor regulating beta-casein gene expression, was elevated in PhIP-exposed HC11 cells. AG490, a Janus kinase 2 (JAK2)-specific inhibitor, blocked the effect of PhIP on beta-casein gene expression. PhIP-treated cells also showed higher expression of Bcl-2 and lower expression of Bax, consistent with a possible antiapoptotic action of PhIP. The findings indicate that PhIP modulates lactogenic hormone-mediated gene expression in mammary epithelial cells, apparently via enhanced phosphorylation of STAT5A. The findings have implications for a novel mechanism of action of the mammary gland carcinogen PhIP.

Lobe-specific increases in malondialdehyde DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus.

Helicobacter hepaticus infection is associated with chronic hepatitis and the development of liver tumours in mice. The underlying mechanism of this liver carcinogenesis is not clear but the oxidative stress associated with H. hepaticus infection may result in induction of lipid peroxidation and the generation of malondialdehyde. Malondialdehyde can react with deoxyguanosine in DNA resulting in the formation of the cyclic pyrimidopurinone N-1,N(2) malondialdehyde-deoxyguanosine (M1dG) adduct. This adduct has the potential to cause mutations that may ultimately lead to liver carcinogenesis. The objective of this study was to determine the control and infection-related levels of M1dG in the liver DNA of mice over time, using an immunoslot-blot procedure. The level of M1dG in control A/J mouse livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 10(8) nucleotides, respectively. Higher levels of M1dG were detected in the liver DNA of H. hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 10(8) nucleotides at 3, 6, 9 and 12 months, respectively. There was a significant age dependent increase in the level of M1dG in the caudate and median lobes of the A/JCr mice relative to control mice. A lobe specific distribution of the M1dG adduct in both infected and control mice was noted, with the left lobe showing the lowest level of the adduct compared with the right and median lobes at all time points. In a separate series of mice experimentally infected with H. hepaticus, levels of 8-hydroxy-deoxyguanosine were significantly greater in the median compared with the left lobe at 12 weeks after treatment. In conclusion, these results suggest that M1dG occurs as a result of oxidative stress associated with H. hepaticus infection of mice, and may contribute to liver carcinogenesis in this model.

Psychological functioning of recently bereaved, middle-aged women: the first 13 months.

This study examined the natural course of psychological functioning in recently bereaved middle-aged women. 69 widows were assessed four times (T1-T4) between the period of 4 to 13 mo. after the loss and were compared to a matched nonwidowed group of 57. Of the SCL-90 feelings of depression, agoraphobic behavior, anxiety, hostility, somatization, feelings of insufficiency, and sleep disorders were heightened at 4 mo. after bereavement compared to the norm group. Significantly higher psychological dysfunctioning was found on all SCL-90 subscales than for non-widows. Over time, a decrease in psychological dysfunction was found for most widows; however, not every widow appeared to recover psychologically, and 17% of the widows showed severe psychological dysfunctioning at 13 mo. postbereavement (T4). With respect to the predictive value of the Total score on the SCL-90, at 13 mo., 27% of these widows had scores indicating severe psychological dysfunctioning; these were comparable to their scores at 4 mo. postbereavement.

Age-related alterations in 32P-postlabeled DNA adducts in livers of mice infected with the tumorigenic bacterial pathogen, Helicobacter hepaticus.

Helicobacter hepaticus causes chronic active hepatitis and liver tumors in mice, with associated increase in reactive oxygen species. Indigenous (I)-compounds are bulky DNA adducts present at low levels and detected by 32P-post-labeling. Some may be caused by reactive oxygen species; others occur normally and decrease during liver tumorigenesis. The identity of most is unknown. We investigated whether mouse liver infection by H. hepaticus and resulting progression of hepatic lesions would be associated with qualitative or quantitative changes in I-compounds. Mice were 3, 6, 9, and 12 months of age; liver disease ranged from minimal through marked. In control A/J mice, up to 20 I-compounds were detected, and the total level of these did not change with age, whereas 11 individual I-compounds showed marked age-related differences. These appeared to be coordinately regulated, as the total of these 11 adducts was constant at 6-12 months. In A/JNCr mice naturally infected with H. hepaticus, up to 12 hepatic I-compounds were found. Total levels varied markedly with age and were high at 6 and 12 months. Neither total adduct levels, nor the amount of any individual adduct, correlated positively with severity of hepatic lesions; in some cases, highest levels were found in livers with least disease. Thus, liver infection and tumorigenesis by H. hepaticus was not associated with an increase in any 32P-postlabeled DNA adduct. Marked, and distinct, age-related changes in total or individual adducts in control and infected mice suggest a role in the physiological alterations of aging and in host response to infection.

Tea as a potential chemopreventive agent in PhIP carcinogenesis: effects of green tea and black tea on PhIP-DNA adduct formation in female F-344 rats.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of proteinaceous animal foods (meat, chicken, and fish). PhIP is a carcinogen in the Fischer 344 (F-344) rat; it induces mammary tumors in female rats and lymphomas and colon and prostate tumors in male rats. In F-344 rats, PhIP forms DNA adducts in various organs, including the target organs. Inhibition of PhIP-DNA adduct formation is likely to lead to inhibition of PhIP tumorigenicity. We have examined the chemopreventive properties of green tea and black tea in PhIP carcinogenesis by evaluating their effects on PhIP-DNA adduct formation in the female F-344 rat. Young adult animals were maintained on powdered AIN-76A diet while receiving regular drinking water or 2% (wt/vol) infusions of green tea or black tea for a total of six weeks. During Weeks 3, 4, and 5, all animals received PhIP by gavage (1 mg/kg/day). Three rats per group were euthanized on Days 1 and 8 after termination of PhIP exposure. DNA was isolated from a number of organs and analyzed for PhIP-DNA adducts by 32P-postlabeling assays. Compared with animals on regular drinking water, PhIP-DNA adduct formation was inhibited in small intestine, colon, liver, and mammary epithelial cells (MECs) of animals receiving green tea or black tea as the sole source of drinking fluid. Green tea inhibited adduct formation in colon, liver, and MECs (33.3-80.0%) on both days, but only on Day 8 (54.4%) in small intestine. Black tea inhibited adduct formation on both days in liver (71.4-80.0%), on Day 1 in colon (40.0%), and on Day 8 in small intestine (81.8%); it had no effect on MEC adducts. Neither green tea nor black tea had an effect on adduct levels in pancreas, lungs, white blood cells, heart, kidneys, spleen, cecum, or stomach. Similarly, these teas did not affect the rate of adduct removal (percent change from Day 1 to Day 8) in any organ. It is concluded that green tea and black tea are potential chemopreventive agents in PhIP-induced tumorigenesis in the F-344 rat.

Association between acetylator genotype and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adduct formation in colon and prostate of inbred Fischer 344 and Wistar Kyoto rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine (HCA) found in cooked meats, causes colon and prostate tumors in male rats. Polymorphic N-acetyltransferase metabolizes N-hydroxy-PhIP to a DNA-reactive form. Liver, colon, and prostate PhIP-DNA adduct levels were compared in male rapid-acetylator Fischer 344 (F344) and slow-acetylator Wistar-Kyoto (WKY) rats fed 0.01 or 0.04% PhIP. Liver PhIP-DNA adduct levels at both PhIP doses, and colon PhIP-DNA adduct levels at the 0.01% PhIP dose were unaffected by acetylator genotype. However, in rats fed 0.04% PhIP, colon PhIP-DNA adduct levels were higher in rapid acetylator F344 rats (P < 0.05). Similarly, prostate PhIP-DNA adduct levels were higher in rapid acetylator F344 rats at both PhIP doses (P < 0.05). The combination of the high-PhIP dose and rapid-acetylator genotype resulted in the highest level of PhIP-DNA adducts in rat colon and prostate.

Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats.

The chemopreventive properties of dietary indole-3-carbinol (I3C) were evaluated by assessing its effect on DNA adduct formation and metabolism of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and the induction of cytochromes P450 1A1 and -1A2 in female F344 rats. In experiment 1, animals on I3C diets (0, 0.02% or 0.1%, w/w) were treated by gavage with 1mg/kg/day of PhIP for 23 days. On days 2, 9, 16 and 23, their 24-hr urine was collected and unmetabolized PhIP was measured by GC/MS. On day 24, the animals were sacrificed, and DNA from pancreas, spleen, white blood cells (WBCs), lung, colon, kidney, mammary epithelial cells, caecum, heart, small intestine, liver and stomach was isolated for determination of PhIP-DNA adduct levels by (32)P-postlabelling assays. Except in the mammary gland, I3C diets significantly inhibited PhIP-DNA adduct formation in WBCs and in all organs, ranging from 34.7 to 67.7% with the 0.02% I3C diet to 68.4 to 95.3% with the 0.1% I3C diet. I3C diets also significantly decreased the concentration of urinary unmetabolized PhIP to 29.5-38.4% (0.02% I3C) and 12.8-17.8% (0.1% I3C) of values obtained with the I3C-free diet. In experiment 2, animals were either treated by intubation of I3C at 100 or 200mg/kg for 2 consecutive days or given an I3C-containing diet (0.02% or 0.1%, w/w) for 2 weeks. The expression and activity of cytochromes P450 1A1 and -1A2 were studied by Northern blots, Western blots, and in vitro enzyme determinations. Both the expression and activity of these cytochromes were induced by all of the I3C treatments. It is concluded that, in the female F344 rat, dietary I3C inhibits PhIP-DNA adduct formation and accelerates PhIP metabolism, probably through induction of cytochromes P450 1A1 and -1A2. The chemopreventive properties of I3C in PhIP-induced carcinogenesis are probably mediated through enhancement of PhIP detoxification pathways.

Inhibition of cell death in human mammary epithelial cells by the cooked meat-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet in cooked meat. To examine if PhIP and its reactive metabolite N-hydroxy-PhIP inhibit apoptosis in human mammary epithelial MCF-10A cells, confluent cultures deprived of serum and growth factors were incubated for 24 h with either compound. The percentages of dead cells (mean +/- SEM, n = 3) as measured by trypan blue exclusion were 5.7 +/- 0.6, 3.4 +/- 0.3, 2.7 +/- 0.3, and 0.2 +/- 0.003%, in control, 1 microM N-hydroxy-PhIP-, 5 microM N-hydroxy-PhIP-, and 100 microM PhIP-treated dishes, respectively. The expression of Bcl-2 and Bcl-x(L) as quantitated by Western blotting was 1.2- to 1.9-fold higher in the treated groups. PhIP-DNA adducts induced by N-hydroxy-PhIP in MCF-10A cells measured by the (32)P-postlabeling assay were low (<1 x 10(7), relative adduct labeling). No adducts were detected after incubation with PhIP. Western blot analysis indicated that PhIP increased ERK2 phosphorylation concomitant with Bcl-2. The results suggest that the inhibition of cell death in mammary epithelial cells by PhIP occurs independently of PhIP-DNA adducts and may involve enhanced signaling through the MAP kinase pathways.

Dietary omega-3 fatty acids as potential inhibitors of carcinogenesis: effect on DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice and rats.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in the CDF1 mouse, causing lymphomas (spleen and lymph nodes) and in the F344 rat, causing mammary tumours in the female and colon tumours in the male. Dietary fish oil, a rich source of omega-3 fatty acids, exhibits chemopreventive properties in several rodent tumour models. The potential chemopreventive properties of dietary omega-3 fatty acid ethyl ester concentrate (O3C) were tested by evaluating its effects on the formation and removal of PhIP-DNA adducts. In the first experiment, a powdered AIN-76A diet containing 4.0% (w/w) O3C inhibited PhIP-DNA adduct formation in various organs of the CDF1 mouse, but not in those of the F344 rat. In a subsequent, second experiment, groups of male CDF1 mice were maintained for 43 days on AIN-76A diets containing the following percentages (w/w) of corn oil ethyl esters and O3C: 7.0 and 0, 5.5 and 1.5, 4.0 and 3.0, and 1.0 and 6.0, respectively. All animals received 0.04% (w/w) PhIP in the diet during weeks 3 and 4. Using 32P-postlabelling assays, PhIP-DNA adducts were analysed in various organs and white blood cells (WBC) on days 1, 8 and 15 after removal of PhIP from the diet. In the liver, O3C-containing diets inhibited adduct formation at all three time points (40.3-60.0%, 53.4-75.7% and 43.3-64.3% on days 1, 8 and 15, respectively). In the spleen, inhibition was evident only on days 8 (35.4-38.8%) and 15 (38.4-56.5%). O3C diets inhibited adduct formation in the stomach, small intestine and caecum at all three time points (except in the stomach and caecum on day 15) amounting to 18.5-31.5% decreases in the stomach, 40.0-60.3% decreases in the small intestine and 24.4-31.4% decreases in the caecum. The extent of inhibition was not related to O3C concentration. In the colon and WBC, adduct levels were independent of the type of diet. In all organs, adduct levels decreased significantly over time, with day 15 levels being 6.3-31.6% of those on day 1. Rate of adduct removal was independent of the type of diet. It is concluded that dietary O3C inhibits PhIP-DNA adduct formation in a target organ (spleen) as well as in non-target organs (liver and gastrointestinal tract) of the CDF1 mouse, but that the rate of adduct removal is independent of the O3C content of the diet.

N-Acetyltransferase expression and DNA binding of N-hydroxyheterocyclic amines in human prostate epithelium.

Intact prostate epithelial cells prepared from benign prostatic hypertrophy tissues from two patients were incubated for 2 h with N-hydroxy derivatives of 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (N-OH-PhIP) or 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline (N-OH-MeIQx). (32)P-post-labeling analysis detected PhIP and MeIQx adducts in the DNA of these cells but not in the untreated control. Adduct levels were approximately 100 times greater in N-OH-PhIP- than in N-OH-MeIQx-treated cells. Repair synthesis of DNA was observed in cells, prepared from two additional patients, treated for 24 h with these carcinogens and was greater for N-OH-PhIP than for N-OH-MeIQx. PhIP, MeIQx and their nitro derivatives did not produce repair synthesis of DNA in this system. The difference in the activity of N-OH-PhIP and N-OH-MeIQx may be due to their stability, since N-OH-MeIQx decomposed rapidly in neutral solution. Transcripts of NAT1 and NAT2 were detected by an in situ hybridization method in prostate epithelial cells, but were absent from stromal tissues. These results suggest that PhIP may be a potential carcinogen for human prostate, since cooked meats, which contain this heterocyclic amine, have been associated with human prostate cancer.

Inhibition of DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline by dietary indole-3-carbinol in female rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) are two important heterocyclic amines formed in proteinaceous foods during the cooking process. Both PhIP and IQ are carcinogenic in several strains of rats. PhIP induces mammary tumors in female F344 rats, while IQ induces principally mammary and liver tumors in female Sprague-Dawley rats. Both PhIP and IQ are activated enzymatically, first by N-hydroxylation, catalyzed by CYP1A1 and CYP1A2, and subsequently by esterification (O-acetylation or sulfation), to yield DNA adducts. Such DNA adduct formation, and persistence of adducts, is related to initiation of carcinogenesis, while inhibition of this process leads to prevention of carcinogenesis. Indole-3-carbinol (I3C), a constituent of cruciferous vegetables, has chemopreventive properties in various systems; it probably acts by induction of detoxification enzymes. We have examined the effect of dietary I3C on DNA adduct formation by PhIP in female F344 rats and on that by IQ in female Sprague-Dawley rats. In experiment 1, F344 rats were maintained on AIN-76A diet containing 0.1% (w/w) I3C and then given p.o. doses (10 or 50 mg/kg) of PhIP. These doses are known to induce CYP1A1 and CYP1A2. Groups of animals (4/time point) were euthanized 1, 2, 6, and 16 days later, and their blood (for isolation of white blood cells), mammary glands, liver, stomach, small intestine, cecum, colon, heart, lungs, kidneys, and spleen were removed for DNA isolation and quantitation of PhIP-DNA adducts by 32P-postlabeling. PhIP-DNA adduct formation was inhibited (40-100%) by I3C in virtually all organs, including the mammary gland (the target organ), at both doses of PhIP, and at almost all time points. In a second experiment, Sprague-Dawley rats were fed either control AIN-76A diet or this diet containing 0.02% I3C or 0.1% I3C for a total of 42 days. IQ was added to the diets (0.01%, w/w) from day 15 to day 42, after which all rats received diet free of IQ and I3C. Groups of animals (4/time point) were killed on days 43 and 57. In addition to the organs removed in experiment 1, the pancreas, uterus, and ovaries were also removed, and IQ-DNA adducts were quantitated by 32P-postlabeling. Both dietary concentrations of I3C inhibited IQ-DNA adduct formation in most organs (except in lungs, kidneys, and pancreas) on both days 43 and 57; in liver, stomach, mammary gland, and spleen, inhibition was evident only on day 43. Inhibitions ranged from 22.6 to 86.6% with the 0.02% I3C diet and from 32.2 to 89.6% with the 0.1% I3C diet. I3C diets did not affect rate of adduct removal in either experiment. It is concluded that dietary I3C inhibits PhIP- and IQ-DNA adduct formation in both target and nontarget organs of female rats, even with high doses of PhIP when CYP1A1 and CYP1A2, the enzymes responsible for the initial activation (N-hydroxylation) of PhIP, are expected to be induced.

In vivo mutagenicity and hepatocarcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in bitransgenic c-myc/lambda lacZ mice.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amine found in cooked meat. Hepatic DNA adduct formation, in vivo mutagenicity, and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene (Muta mice) and bitransgenic mice overexpressing the c-myc oncogene. C57Bl/lambda lacZ and bitransgenic c-myc (albumin promoter)/lambda lacZ mice were bred and weaned onto an American Institute of Nutrition-76-based diet containing 0.06% (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma (100% incidence). By 40 weeks, hepatic tumor incidence was 100%/75% (17%/0%) and 44%/17% (0%/0%) in male c-myc/lambda lacZ and C57Bl/lambda lacZ mice who were given MeIQx (or control) diet, respectively, supporting a synergism between MeIQx and c-myc overexpression in hepatocarcinogenesis. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts, as detected by the 32P-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alterations were base substitutions at guanine bases. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a 1.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/lambda lacZ and C57Bl/lambda lacZ mice after 30 weeks on diet. Thus, it seemed that factors in addition to MeIQx-DNA adduct levels, such as the enhanced rate of proliferation associated with c-myc overexpression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/lambda lacZ mice than in C57Bl/lambda lacZ mice. The findings are consistent with the notion that c-myc overexpression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc overexpression on MeIQx hepatocarcinogenicity seems to involve an enhanced expression of MeIQx-induced mutations.

DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis.

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds produced during the pyrolysis of creatine, amino acids and proteins. The major subclass of HCAs found in the human diet comprise the aminoimidazoazaarenes (AIAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All, except DiMeIQx, have been shown to be carcinogenic in animals. These compounds are present in cooked muscle meats at the p.p.b. level. Since the discovery of the HCAs in the late 1970s, many studies have examined the DNA adducts of these compounds. This review compiles the literature on AIA-DNA adducts including their identification and characterization, pathways of formation, mutagenesis in vitro and in vivo, and their association with carcinogenesis in animal models. It is now known that metabolic activation leading to the formation of DNA adducts is critical for mutagenicity and carcinogenicity of these compounds. All of the AIAs studied adduct to the guanine base, the major adduct being formed at the C8 position. Two AIAs, IQ and MeIQx, also form minor adducts at the N2 position of guanine. A growing body of literature has reported on the mutation spectra induced by AIA-guanine adducts. Studies of animal tumors induced by AIAs have begun to relate AIA-DNA adduct-induced mutagenic events with the mutations found in critical genes associated with oncogenesis. Several studies have demonstrated the feasibility of chemoprevention of AIA tumorigenesis. Only a few studies have reported on the detection of AIA-DNA adducts in human tissues; difficulties persist in the routine detection of AIA-DNA adducts in humans for the purpose of biomonitoring of exposure to AIAs. The AIAs are nevertheless regarded as possible human carcinogens, and future research on AIA-DNA adducts is likely to help address the role of AIAs in human cancer.

Prevention by aspirin and its combination with alpha-difluoromethylornithine of azoxymethane-induced tumors, aberrant crypt foci and prostaglandin E2 levels in rat colon.

The dose-response relationship in male F344 rats was determined for the ability of aspirin administered in the diet to prevent azoxymethane (AOM)-induced colon cancer and aberrant crypt foci (ACF) and to reduce prostaglandin E2 (PGE2) levels. Starting at either 7 or 22 weeks of age, the rats received aspirin. All rats received two doses of AOM (15 mg/kg each on days 7 and 14) and were killed on day 36. The lowest concentrations of aspirin to prevent ACF or reduce PGE2 levels were 600 and 400 mg/kg, respectively. To evaluate the prevention of tumors, rats received either 0 or 400 mg/kg aspirin for a total of 39 weeks with AOM (30 mg/kg) administered 7 days after the start of treatment. Aspirin had no effect on the yield of colon tumors. In a second experiment, rats started to receive 0, 200, 600 or 1800 mg/kg aspirin or 1000 mg/kg alpha-difluoromethylornithine (DFMO) +/- aspirin. Eight and 15 days later, all the rats received 15 mg/kg AOM. Eleven weeks later, animals that were receiving the control diet started to receive 0, 200, 600 or 1800 mg/kg aspirin; 1000 or 3000 mg/kg DFMO; or 1000 mg/kg DFMO + 200 or 600 mg/kg aspirin. The animals were killed 32 weeks later. DFMO effectively reduced the yield of colon tumors when administered starting either before or after AOM while aspirin was much weaker. The combination of aspirin + DFMO administered after AOM was synergistic. Both aspirin and DFMO decreased the Mitotic Index, while apoptosis was increased only by DFMO. Our results demonstrated that aspirin and DFMO could prevent colon cancer when administered after AOM. Furthermore, aspirin reduced ACF, PGE2 levels and mitosis at concentrations that did not prevent cancer. In contrast, the ability to enhance apoptosis did correlate with the prevention of cancer.

The immunological and psychological effects of bereavement: does grief counseling really make a difference? A pilot study.

This study evaluates psychological and immunological functioning after bereavement and the influence of group counseling. Eighteen widows (bereaved within 3 months of enrolment) and a reference group of 10 married control subjects were asked to fill in self-report scales and to donate a blood sample (T1). After T1, half of the widows (the experimental group) were randomly assigned to grief counseling (13 sessions over 4 months), while the other subjects (the control group) received no treatment. Seven months after bereavement (T2) or, in the case of the experimental group, immediately after the intervention, a follow-up was conducted in the widowed subsample using the same measures. Blood samples were analyzed to determine the total number of white blood cells, number of lymphocyte subsets, natural killer cell activity (NKCA) and lymphocyte proliferative response to phytohemagglutinin (PHA), anti-CD3 and pokeweed mitogen (PWM). At T1, we found significant differences between widows and non-widows regarding both psychological and immunological measures. Widows felt more anxious, depressed, hostile and agoraphobic. At T1, widows had a lower number of the CD19+CD5+ B cell subpopulation. The cell function tests for T and B cells showed higher responses in widows (lymphocyte proliferation response to PHA, anti-CD3 and PWM). No significant difference in NKCA was found between widows and non-widows. At T2, there appeared to be no significant difference between widows and non-widows on the psychological measures. With respect to the immunological measures, widows and non-widows showed no significant differences for the total number of white blood cells, number of lymphocyte subsets and NKCA. Consistent with our findings at T1, the lymphocyte proliferation response to PHA, anti-CD3 and PWM at T2 appeared to be higher in widows than in non-widows. Comparing the experimental group (widows) and the control group (widows) with respect to psychological measures at T1, widows in the experimental group felt more insufficient and had more sleep disturbances. With respect to the immunological measures, no differences were found between those two groups. When the same two groups were again compared at T2, no differences were found in any of the psychological or immunological measures (lymphocyte sub-populations, proliferation tests and the NKCA).

Formation of DNA adducts of the food-derived mutagen 2-amino-9H-pyrido-[2,3-b]indole (A(alpha)C) and bioassay of mammary gland carcinogenicity in Sprague-Dawley rats.

2-amino-9H-pyrido[2,3-b]indole (AalphaC) is a heterocyclic amine found at relatively high concentrations in barbecued or grilled meats. In the current study, the mammary gland carcinogenicity of AalphaC was examined in female Sprague-Dawley rats given 10 doses of AalphaC (75 mg/kg, orally, once per day starting at 43 days of age) and placed on a defined high-fat diet (23.5% corn oil), a strong promotional factor for rat mammary gland carcinogenesis. Within 1 year, one out of 20 rats dosed with AalphaC developed a tubulopapillary carcinoma, indicating that the bioassay was largely negative. As DNA adduct formation is considered to play a role in carcinogenesis, AalphaC-DNA adduct levels were measured in the mammary gland and other tissues by the 32P-postlabelling method. Under intensification conditions, one major adduct and up to three minor adducts were detected in isolated mammary gland epithelial cells and other tissues (liver, stomach, small intestine, colon and kidney) of AalphaC-treated rats; the adduct patterns were similar in all tissues examined. The major adduct, comprising 60-100% of total DNA adduct levels in tissues, was chromatographically identical to the principal adduct found in 3'-dGp-AalphaC (synthesized by reacting 3'-phospho-2'-deoxyguanosine (3'-dGp) with N-acetoxy-AalphaC). Of the tissues examined, the highest AalphaC-DNA adduct levels were found in the liver. In male rats given a single dose of AalphaC (75 mg/kg, orally, 3 hr prior to necropsy), no AalphaC-DNA adducts were detected in extrahepatic tissues. In female rats given a single dose or 12 daily doses of AalphaC, hepatic DNA adduct levels were at least 12-13-fold higher than those in any other tissue. Mean total AalphaC-DNA adduct levels in mammary gland epithelial cells and liver from female rats given multiple doses of AalphaC were 3.5 and 50.7 (RAL x 10(7)), respectively. Although factors in addition to DNA adduct formation are likely to play a role in mammary gland carcinogenesis, the results suggest that the weak mammary gland carcinogenicity of AalphaC may in part be associated with low AalphaC-DNA adduct levels in the mammary gland epithelium.

Proliferation, development and DNA adduct levels in the mammary gland of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a high fat diet.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine derived from cooked meat that is a mammary gland carcinogen in rats. A carcinogenic dose-regimen of PhIP (75 mg/kg, p.o., 10 doses, once per day) was administered to 43-day old female Sprague-Dawley rats, and the rats were then placed on a defined high fat (23.5% corn oil) or low fat (5% corn oil) diet for up to 6 weeks. At various times after carcinogen and diet, and prior to carcinogenesis, we examined the percentage of proliferating cells in terminal end bud (TEB) epithelial structures of the rat mammary gland by proliferating cell nuclear antigen staining, mammary gland architecture by whole mounting, and PhIP-DNA adduct levels in mammary epithelial cells by the 32P-post-labeling assay. Immediately after dosing, the percentage of proliferating epithelial cells in TEBs was significantly higher in PhIP-treated rats than in control rats receiving vehicle only [7.5 +/- 0.9% (n = 99) versus 4.2 +/- 0.6% (n = 127), respectively]. The mammary glands of PhIP-treated rats showed a significantly lower density of alveolar buds (ABs) and a higher density of TEBs than control rats, which suggests that PhIP exposure partially inhibited the normal glandular differentiation of TEBs to ABs. After 6 weeks on the diet, proliferation in TEBs was statistically higher in rats given PhIP plus a high fat diet than in rats given vehicle plus a low fat diet. The mammary glands from rats on a high fat diet also showed a statistically higher density of TEBs when compared with rats on a low fat diet [2.08 +/- 0.34% versus 1.04 +/- 0.20%, respectively (n = 6)]. PhIP-DNA adduct levels were relatively high in mammary epithelial cells of treated rats. At 3 h after the last dose of PhIP, DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SE] were 10.5 +/- 1.7 (n = 8) and 0.9 +/- 0.2 (n = 7) in epithelial cells isolated from mammary gland and in the liver, respectively. DNA adduct removal rates from the mammary gland were not different between rats on the high fat and low fat diets. Adducts were still detected after 6 weeks on either diet. Thus, events that occurred prior to neoplasia in the mammary glands of PhIP-treated rats include formation of PhIP-DNA adducts at relatively high levels, and enhanced proliferation in TEBs (putative sites of origin of mammary gland carcinomas) and partial inhibition of TEB differentiation. The high fat diet, a promoter of PhIP-induced mammary gland carcinogenesis, appeared to sustain the proliferative effect of PhIP in mammary gland TEBs at a time when PhIP-DNA adducts are still detectable. These early events may contribute to the targeting and carcinogenicity of PhIP to the mammary gland of rats.