Dan Segerbäck

PRIMA-1 Reactivates Mutant p53 by Covalent Binding to the Core Domain

Restoration of wild-type p53 expression triggers cell death and eliminates tumors in vivo. The identification of mutant p53-reactivating small molecules such as PRIMA-1 opens possibilities for the development of more efficient anticancer drugs. Although the biological effects of PRIMA-1 are well demonstrated, little is known about its molecular mechanism of action. We show here that PRIMA-1 is converted to compounds that form adducts with thiols in mutant p53. Covalent modification of mutant p53 per se is sufficient to induce apoptosis in tumor cells. These findings might facilitate the design of more potent and specific mutant p53-targeting anticancer drugs.

Birth Weight, Head Circumference, and Prenatal Exposure to Acrylamide from Maternal Diet: The European Prospective Mother–Child Study (NewGeneris)

Background: Acrylamide is a common dietary exposure that crosses the human placenta. It is classified as a probable human carcinogen, and developmental toxicity has been observed in rodents. Objectives: We examined the associations between prenatal exposure to acrylamide and birth outcomes in a prospective European mother–child study. Methods: Hemoglobin (Hb) adducts of acrylamide and its metabolite glycidamide were measured in cord blood (reflecting cumulated exposure in the last months of pregnancy) from 1,101 singleton pregnant women recruited in Denmark, England, Greece, Norway, and Spain during 2006–2010. Maternal diet was estimated through food-frequency questionnaires. Results: Both acrylamide and glycidamide Hb adducts were associated with a statistically significant reduction in birth weight and head circumference. The estimated difference in birth weight for infants in the highest versus lowest quartile of acrylamide Hb adduct levels after adjusting for gestational age and country was –132 g (95% CI: –207, –56); the corresponding difference for head circumference was –0.33 cm (95% CI: –0.61, –0.06). Findings were similar in infants of nonsmokers, were consistent across countries, and remained after adjustment for factors associated with reduced birth weight. Maternal consumption of foods rich in acrylamide, such as fried potatoes, was associated with cord blood acrylamide adduct levels and with reduced birth weight. Conclusions: Dietary exposure to acrylamide was associated with reduced birth weight and head circumference. Consumption of specific foods during pregnancy was associated with higher acrylamide exposure in utero. If confirmed, these findings suggest that dietary intake of acrylamide should be reduced among pregnant women.

Transplacental transfer of acrylamide and glycidamide are comparable to that of antipyrine in perfused human placenta

Most drugs can penetrate the placenta but there are only a few studies on placental transfer of environmental toxic compounds. In this study, we used dual recirculating human placental perfusion to determine the transfer rate through the placenta of a neurotoxic and carcinogenic compound found in food, acrylamide and its genotoxic metabolite glycidamide. Putative acrylamide metabolism into glycidamide during the 4-h perfusions and acrylamide-derived DNA adducts in placental DNA after perfusions were also analyzed. Placentas were collected immediately after delivery and kept physiologically functional as confirmed by antipyrine kinetics, glucose consumption and leak from fetal to maternal circulation. Acrylamide (5 or 10 microg/ml) or glycidamide (5 microg/ml), both with antipyrine (100 microg/ml), was added to maternal circulation. Acrylamide and glycidamide were analyzed in the perfusion medium by liquid chromatography/mass spectrometry. Acrylamide and glycidamide crossed the placenta from maternal to fetal circulation with similar kinetics to antipyrine, suggesting fetal exposure if the mother is exposed. The concentrations in maternal and fetal circulations equilibrated within 2h for both studied compounds and with both concentrations. Acrylamide metabolism into glycidamide was not detected during the 4-h perfusions. Moreover, DNA adducts were undetectable in the placentas after perfusions. However, fetuses may be exposed to glycidamide after maternal metabolism. Although not found in placental tissue after 4h of perfusion, it is possible that glycidamide adducts are formed in fetal DNA.

Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide: quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabelling

7-(2-Hydroxypropyl)guanine (7-HPG) constitutes the major adduct from alkylation of DNA by the genotoxic carcinogen, propylene oxide. The levels of 7-HPG in DNA of various organs provides a relevant measure of tissue dose. 7-Alkylguanines can induce mutation through abasic sites formed from spontaneous depurination of the adduct. In the current study the formation of 7-HPG was investigated in male Fisher 344 rats exposed to 500 ppm of propylene oxide by inhalation for 6 h/day, 5 days/week, for up to 20 days. 7-HPG was analyzed using the 32P-postlabelling assay with anion-exchange cartridges for adduct enrichment. In animals sacrificed directly following 20 days of exposure, the adduct level was highest in the respiratory nasal epithelium (98.1 adducts per 10(6) nucleotides), followed by olfactory nasal epithelium (58.5), lung (16.3), lymphocytes (9.92), spleen (9.26), liver (4.64), and testis (2.95). The nasal cavity is the major target for tumor induction in the rat following inhalation. This finding is consistent with the major difference in adduct levels observed in nasal epithelium compared to other tissues. In rats sacrificed 3 days after cessation of exposure, the levels of 7-HPG in the aforementioned tissues had, on the average, decreased by about one-quarter of their initial concentrations. This degree of loss closely corresponds to the spontaneous rate of depurination for this adduct (t 1/2 = 120 h), and suggests a low efficiency of repair for 7-HPG in the rat. The postlabelling assay used had a detection limit of one to two adducts per 10(8) nucleotides, i.e. it is likely that this adduct could be analyzed in nasal tissues of rats exposed to less than 1 ppm of propylene oxide.

Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark.

The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography-mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/maternal hemoglobin adduct level ratios were 0.48 (range 0.27-0.86) for acrylamide, 0.38 (range 0.20-0.73) for glycidamide, and 0.43 (range 0.17-1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38-0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large population studies.

Placental transfer and DNA binding of benzo(a)pyrene in human placental perfusion

Benzo(a)pyrene (BP) is the best studied polycyclic aromatic hydrocarbon, classified as carcinogenic to humans. The carcinogenic metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), binds covalently to DNA. The key enzyme in this metabolic reaction is CYP1A1, which has also been found in placenta and human trophoblastic cells. By using human placental perfusion we confirmed that BP added to the maternal circulation in concentrations of 0.1 and 1 microM reaches fetal compartment but somewhat slower than the freely diffusible reference substance antipyrine. A well-known P-glycoprotein (ABCB1/P-gp) antagonist verapamil did not affect the transfer more than it did in the case of antipyrine, indicating that ABCB1/P-gp does not have a role in BP transfer. In one of the two placentas perfused for 6 h with the higher concentration of BP (1 microM) BPDE specific DNA adducts were found in placental tissue after the perfusion, but not before. The ability of human trophoblastic cells to activate BP to BPDE-DNA adducts was confirmed in human trophoblastic BeWo cells. This study shows that maternal exposure to BP leads to the exposure of the fetus to BP and/or its metabolites and that placenta itself can activate BP to DNA adducts.

Propylene oxide: mutagenesis, carcinogenesis and molecular dose.

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO-DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC-HRMS) analysis. The second method utilized 32P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4 +/- 80.1 (respiratory), 396.8 +/- 53.1 (olfactory) and 34.6 +/- 3.0 (liver) pmol adduct/mumol guanine using GC-HRMS. Lower values, 592.7 +/- 53.3, 296.5 +/- 32.6 and 23.2 +/- 0.6 pmol adduct/mumol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by 32P-postlabeling yielded the following values: 445.7 +/- 8.0 (respiratory), 301.6 +/- 49.2 (olfactory) and 20.6 +/- 1.8 (liver) pmol adduct/mumol guanine in DNA of rats killed immediately after exposure cessation and 327.1 +/- 21.7 (respiratory), 185.3 +/- 29.2 (olfactory) and 15.7 +/- 0.9 (liver) pmol adduct/mumol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide.

Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+/-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the (32)P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.

Transplacental Transfer of Nitrosodimethylamine in Perfused Human Placenta

Nitrosodimethylamine (NDMA) is a carcinogenic compound present in tobacco smoke and food such as cured meat, smoked fish and beer. The O(6)-methylguanine formed in human cord blood in mothers highly exposed to such products implicates NDMA exposure of the fetus. Dual recirculating human placental perfusion was used to get direct evidence of the transplacental transfer of NDMA and DNA adduct formation in perfused human placenta. Eleven placentas from normal full-term pregnancies were collected immediately after delivery and an isolated lobule was perfused with 1 or 5 microM of (14)C-NDMA with a reference substance, antipyrine (0.1mg/ml) added to the maternal circulation. Perfusate samples were collected from both maternal and fetal circulations every half an hour for the first two hours and once per hour from thereon. NDMA was analyzed by scintillation counting and antipyrine by high performance liquid chromatography. The transfer of NDMA was comparable to that of antipyrine and probably occurred through passive diffusion, with the concentrations in maternal and fetal sides equilibrating in 2-3h. No indication of any effect by efflux transporters on NDMA kinetics was noticed in the experiments utilizing Caco-2 or MDCK- MDCKII-MDR1 cell culture monolayer in a transwell system, either. Furthermore, no NDMA-DNA-adducts were found after the perfusions and no DNA-binding of NDMA was seen in in vitro incubations with human placental microsomes from 8 additional placentas. Thus, our study demonstrates that the human fetus can be exposed to NDMA from the maternal circulation. According to this study and the literature, NDMA is not metabolized in full-term human placenta from healthy non-smoking, non-drinking mothers. It remains to be studied whether NDMA concentrations high enough to evoke fetal toxicity can be obtained from dietary sources.

Adducts with haemoglobin and with DNA in epichlorohydrin-exposed rats.

Epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) is an important industrial chemical and a carcinogen in experimental animals. The main aims of the present study were to characterize the adduct formation in female Wistar rats and to identify adducts that could potentially be used in human biomonitoring studies. The total binding of radioactivity to haemoglobin in rats administered 0, 0. 11, 0.22, 0.43, or 0.97 mmol [3H]ECH/kg body weight by i.p. injection, and sacrificed 24 h after treatment, was linearly related to a dose up to 0.43 mmol/kg body weight. The binding at the highest dose was higher than predicted by extrapolation from lower doses, indicating saturation of a metabolic process for elimination of ECH. Ion-exchange chromatography of a globin hydrolysate showed one major radioactivity peak corresponding to S-(3-chloro-2-hydroxypropyl)cysteine. The half-life of this adduct was estimated as about 4 days by analysis of globin from rats administered 0.43 mmol/kg body weight and sacrificed after 1, 2 and 9 days. Crosslinking of the adduct, presumably with glutathione, appeared to be the predominant secondary reaction. Hydrolysis of N-(3-chloro-2-hydroxypropyl)valine, the primary reaction product of ECH with N-terminal valine, would give N-(2,3-dihydroxypropyl)valine. A sensitive gas chromatography/mass spectrometry method for the dihydroxypropyl adduct was used to follow its formation and removal after administration of nonlabelled ECH (0.11 mmol/kg body weight). The level of this adduct reached a maximum of about 20 pmol/g globin after a few weeks, corresponding to about 0.1% of the initial binding of ECH to globin. N-7-(3-Chloro-2-hydroxypropyl)guanine was detected in rats administered 0.97 mmol [3H]ECH/kg body weight and sacrificed 6 h after treatment. The adduct levels in haemoglobin and DNA were compared with previously reported adduct levels in male Fischer 344 rats exposed to propylene oxide. Despite its higher chemical reactivity, the capacity of ECH to alkylate macromolecules in vivo was found to be somewhat lower than that of propylene oxide.