Effects of dietary transition metals on oxidative DNA lesions in neonatal rats

Bulky endogenous oxidative lesions (type II I-compounds) reflect DNA damage associated with oxidative stress. As shown by 32P-postlabeling, their levels are enhanced by pro-oxidant genotoxins and also shortly after normal birth in several rat tissues as a function of time and the maternal diet. In order to elucidate which dietary components contribute to postnatal DNA damage, we have focused, herein, on the possible role of transition metals (iron, copper, and nickel). Pregnant Fischer 344 (F344) rats were fed AIN-93G purified diet containing different amounts of iron, copper, and nickel, or Purina-5001 natural-ingredient diet (which contains relatively high concentrations of these metals). Type II I-compounds were estimated by nuclease P1-enhanced 32P-postlabeling in liver and lung DNA of fetuses and at 24h and day 9 post-partum. Increased postnatal oxidative damage was detected in liver but not lung DNA of neonates exposed to higher amounts of dietary transition metals. There were significant positive linear correlations between maternal transition metal intake and neonatal, but not fetal and maternal type II I-compound levels. The results show that transition metals in the maternal diet affect perinatal oxidative DNA damage, presumably via a Fenton-type reaction. They also provide evidence for optimal levels in the maternal diet of transition metals, which on one hand, are essential for life, but on the other, can cause potentially deleterious DNA alterations in the offspring.     

Circadian rhythm of covalent modifications in liver DNA.

32P-postlabeling analysis recently revealed that in addition to 5-methylcytosine, mammalian DNA contains covalently modified nucleotides of unknown structures and functions termed I-compounds whose levels increase with age. I-compound levels, in addition, depend on species, strain, sex, tissue, and diet and are generally lowered by carcinogen exposure. As shown here, levels of several non-polar I-compounds in liver DNA of untreated male C3H mice were elevated 2 to 8.5 times at 1800 h and 2400 h as compared to 0600 h and 1200 h, while polar I-compounds and persistent carcinogen-DNA adducts induced by safrole were unaffected by time of day. In liver DNA of male F-344 rats 4 non-polar I-compounds and 4 polar I-compounds showed significant circadian rhythm at 2000 h compared to 0800 h. This novel circadian variation of DNA structure implies mechanisms precisely regulating I-compound levels in vivo and may conceivably be linked to diurnal differences of DNA synthesis and gene expression.     

Bulky endogenous DNA modifications (I-compounds) -possible structural origins and functional implications

I-compounds are bulky covalent DNA modifications which increase with age in tissues of unexposed laboratory animals and are derived from endogenous DNA-reactive intermediates of nutrient and oxygen metabolism. They have been classified into 2 major groups, i.e., type I and type II. Profiles and levels of type I I-compounds show considerable variation depending on species, strain, tissue, and gender, but are also affected by diet and chemical and hormonal exposures, indicating their formation to be determined by genetic and environmental factors. For example, sex hormones, dietary oat lipids, and isoprenoids affect their profiles and/or levels in tissue DNA. A gradual depletion of many type I I-compounds occurs during carcinogenesis, as many carcinogens/tumor promoters significantly reduce their levels, and neoplasms display very low levels, apparently independent of growth rate, indicating a loss of the ability to form these modified nucleotides. Conversely, dietary restriction, the most effective method to retard carcinogenesis and aging, significantly elevates type I I-compound levels, as compared to age-matched ad libitum-fed animals. Levels of many liver and kidney I-compounds exhibit genotype- and diet-dependent positive linear correlations with median life span. Formation of high levels of oat-related type I I-compounds has been associated with reduced formation of carcinogen-induced preneoplastic hepatic foci. These results suggest that such DNA modifications may not represent DNA lesions but may rather be functionally important. This view is supported by circadian rhythms displayed by some I-compounds. Thus, certain type I I-compounds may play a protective role against carcinogenesis and age-associated degenerative processes. Type II I-compounds, on the other hand, represent DNA damage and include several bulky lesions, which are enhanced by pro-oxidant carcinogens such as ferric nitrilotri- acetate (Fe-NTA) in target organ (kidney) DNA of rodents and are identical to products generated by oxidizing DNA or oligonucleotides under Fenton reaction conditions in vitro. Some of these products appear to be base-base or base-sugar intrastrand crosslinks. Notably, Fe-NTA reduces the levels of type I I-compounds in renal DNA. Type II I-compound levels are increased in tissue DNA of normal newborn rats. The formation of oxidative DNA lesions in neonates is most likely caused by oxidative stress associated with the sudden increase of partial oxygen pressure in arterial blood and tissues at birth. In view of the rapid cell replication at this developmental stage, endogenous oxidative DNA lesions sustained early in life may contribute to the development of cancer and degenerative diseases later in life.     

Modulation of DNA modification (I-compound) levels in rat liver and kidney by dietary carbohydrate, protein, fat, vitamin, and mineral content.

I-compounds are DNA modifications detected by 32P-postlabeling that increase with age in rodents without known carcinogen exposure. Diet type (natural ingredient versus purified) greatly influences patterns and levels of I-compounds. To test the hypothesis that I-compound formation is affected, also, by dietary macro- and micronutrients, effects of carbohydrate, protein, fat, vitamin, and mineral content on rat liver and kidney I-compounds were determined. Female Sprague-Dawley rats were fed basic or modified AIN-76A purified diets for 3-6 months. High protein (HP) diet (50%, w/w) increased I-compound levels in liver but not kidney. High carbohydrate (HC) diet (78%) produced a significant increase in the polar as well as total I-compound levels in both tissues. High fat diets (20%) elicited significantly lower levels of liver I-compounds than HC, HP, and basic diets. There were few significant differences between high polyunsaturated (safflower oil) and saturated fat (lard) diet groups. No qualitative differences in I-compound profiles were observed in either tissue. In rats fed basic diet supplemented with vitamins and/or minerals, increased vitamin content reduced the levels of polar I-compounds in liver. No extra diet-induced adducts were observed; all effects were of a quantitative nature. These data provide direct evidence that nutrients significantly influence I-compound levels and support the hypothesis that normal metabolism of nutrients leads to the production of small amounts of DNA-reactive electrophiles. These observations suggest a novel mechanism where nutrient composition of the diet may play a role in development of neoplasia and other adverse health effects.     

Acute and long-term effects of carbon tetrachloride on DNA modifications (I-compounds) in male mouse liver

I-compounds are recently discovered species and tissue dependent covalent DNA modifications which are detectable by the 32P-postlabeling assay for DNA adducts and tend to increase with the animal's age. The effects of the hepatocarcinogen carbon tetrachloride (CCl4) on hepatic I-compounds were studied in 10-12-month-old male ICR mice using the 32P-postlabeling assay. CCl4 was dissolved in corn oil (20%, v/v) and intraperitoneally (i.p.) injected in doses of 0.75 ml/kg (0.375 ml/100 g body weight, 20% CCl4 in corn oil) while control mice received corn oil only (0.375 ml/100 g body wt). Twenty-four h after a single injection of CCl4, the intensity of non-polar I-spots in liver DNA was significantly increased as compared with corn oil treated controls, while the level of one polar I-compound was reduced at 24 h. DNA synthesis (as indicated by [3H]thymidine incorporation) was not significantly affected at 24 h after a single dose of CCl4. To study the long-term effects of CCl4, five groups of mice were given two consecutive weekly injections of 0.75 ml/kg CCl4 (as above) and were sacrificed 1, 4, 8, 12 and 22 weeks after the second treatment. In these groups the total liver I-compound levels were reduced to 17.3-49.0% compared with corresponding controls. The maximum decline was observed at 4 weeks (17.3% of control). Comparison of thymidine incorporation showed no significant increase between control and treated liver DNAs at 1, 4 and 8 weeks after CCl4, suggesting that the decrease in I-compound levels was probably not a secondary effect of increased DNA synthesis during postnecrotic proliferation. Even though there was a trend of recovery between 8 and 22 weeks, I-compound levels still remained significantly lower at 22 weeks (49.0%). Since I-compounds appear to be normal DNA modifications, the results suggest that persistent reduction of I-compound levels contributes to the hepatocarcinogenic effect of CCl4.     

Age-related DNA modifications (I-compounds): Modulation by physiological and pathological processes

I-compounds are covalent DNA modifications that can be detected and measured by ³²P-postlabeling assay because of their DNA-adduct like properties. They accumulate in an age-dependent, highly reproducible manner in tissue DNA of untreated animals in the absence of exogenous carcinogens and, therefore, appear to arise via the interaction of DNA with endogenous reactants formed in the course of normal metabolism. Chromatographically, they exhibit a wide range of polarities, indicative of structural diversity. In addition to age-dependent increases, I-compound profiles exhibit prominent species-, sex-, tissue- and diet-dependent qualitative and quantitative differences. Natural-ingredient (chow) diets produce qualitative differences as well as substantially higher I-compound levels in rat liver and kidney, when compared with purified diets. Modified purified diets containing high carbohydrate, protein, or fat concentrations further modulate I-compound profiles. During liver regeneration, I-compounds behave like DNA adducts rather than m⁵C in that their levels are not quickly restored. Treatment of rats with the hepatocarcinogens 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CCl₄, and peroxisome proliferators as well as with a choline-devoid hepatocarcinogenic diet depressed the age-related increases of I-compound levels in liver, the target organ. Additional ³²P-labeled derivatives were observed only with the peroxisome proliferators and presumably represent DNA adducts of exogenous origin. No I-compounds were detected in a series of Morris hepatomas with different degrees of differentiation. Thus, loss of I-compounds may be associated with altered gene expression/dedifferentiation. On the other hand, the age-dependent accumulation of I-compounds and their adduct-like character suggest potential relations to aging-associated dysdifferentiation and initiation of cancer. Structural complexity indicates different biological roles of I-compounds.     

Age-dependent covalent DNA alterations (I-compounds) in rodent tissues: species, tissue and sex specificities

I-compounds are non-polar covalent DNA modifications of as yet undetermined structure that tend to accumulate in an age-dependent manner in tissues of untreated animals. They are detectable by 32P-postlabeling assay because of their adduct-like properties and chromatographically resemble DNA nucleotides containing bulky/hydrophobic moieties. To determine which factors may be involved in their formation, I-compounds were examined by 32P-postlabeling in liver and kidney DNA of female and male Sprague-Dawley rats and Syrian hamsters of different ages (1, 4 and 10 months and 1, 2.5 and 9.5 months, respectively). The following results were obtained: (i) Every tissue DNA studied contained characteristic I-compounds. (ii) Patterns and amounts of I-compounds were reproducible among animals of the same kind. (iii) There were pronounced organ and species differences. (iv) I-compound patterns were sex-dependent. (v) I-compound levels increased with age in all tissues studied, except in male hamster kidney, a target organ of estrogen-induced carcinogenesis. The highest levels were observed in liver and kidney of 10-month-old female rats. (vi) The rise of I-compound levels was less steep during the later part of the observation period for female but not male animals. (vii) Gonadectomy decreased I-compound levels in female hamster kidney DNA, while causing a slight increase in male animals later in life. These I-compounds were identical to previously reported DNA modifications that increased in male hamster kidneys after prolonged estrogen treatment. Points, iv, vi and vii strongly implicated sex hormones in I-compound formation. The qualitative effects of species, tissue differentiation, gender and sex hormones on these DNA modifications support the hypothesis that I-compounds are formed by the binding of endogenous electrophiles to DNA. As persistent DNA alterations, they are likely to affect DNA replication and to play a role in spontaneous and chemically induced carcinogenesis and in aging.     

A 32P-postlabeling assay for the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine in mammalian tissues: evidence that four type II I-compounds are dinucleotides containing the lesion in the 3' nucleotide.

8,5'-Cyclopurine-2'-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of (32)P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5'-cyclo-2'-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic (32)P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The (32)P-postlabeling technique used here is capable of detecting 1-5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.     

Effects of diet on biomarkers of exposure and effects, and on oxidative damage

Modification of DNA is believed to be a key step in carcinogenesis, and therefore DNA adducts have been proposed as predictive biomarkers of human cancer. Smoked and grilled foods are important contributors of PAH-DNA adduct levels, while the consumption of flavonoids and other antioxidants seems to decrease the level of DNA adducts. The aim of this study was to assess the effect of each group of foods and of different dietary profiles on the DNA adducts levels and on oxidative damage to DNA. Occupationally exposed men were recruited in Czech Republic, Slovak Republic and Bulgaria. Non-occupationally exposed subjects were matched on age and gender to PAH-exposed workers. Three hundred and fifty-six subjects who completed the questionnaire for dietary information and had a measurement of DNA adduct levels and oxidative damage to DNA were included in this study. No food item seemed to be individually associated with markers of exposure or DNA damage. Total DNA adducts levels were significantly higher for subjects who had eaten, in the previous 24 h, smoked or fried food. A Principal Components Analysis was performed to identify groups of subjects with similar dietary profiles: no significant differences in biomarker levels were observed among the groups defined according to dietary profiles. In conclusion, this study did not show any significant association between diet and biomarkers of DNA damage, oxidative damage to DNA and chromosomal aberrations, neither when each food was considered separately, nor when the effect of different dietary profiles was tested. The recent consumption of smoked or fried food was associated with an increase in total DNA adducts levels.     

Maternal selenium deficiency increases hydrogen peroxide and total lipid peroxides in porcine fetal liver

To investigate the role of selenium (Se) in the developing porcine fetus, prepubertal gilts (n=42) were randomly assigned to either Se-adequate (0.39 ppm Se) or Se-deficient (0.05 ppm Se) gestation diets 6 wk prior to breeding. Maternal and fetal liver was collected at d 30, 45, 70, 90, and 114 of pregnancy. Concentrations of Se in maternal liver decreased during gestation in gilts fed the low-Se diet. The activity of cellular glutathione peroxidase (GPx) was decreased at d 30 and 45 of gestation in liver of gilts fed the low-Se diet. Concentrations of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were greater in liver homogenates from gilts fed the low-Se diet. Within the fetuses, liver Se decreased in those fetuses of gilts fed the low-Se diet. Although the activity of GPx in fetal liver was not affected by the maternal diet, concentrations of H₂O₂ and MDA in fetal liver were greater in fetuses from gilts fed the low-Se diet. Maternal liver GPx activity was approx 12-fold greater than fetal liver GPx activity regardless of dietary treatment. These results indicate that maternal dietary Se intake affects fetal liver Se concentration and feeding a low-Se diet during gestation increases oxidative stress to the fetus, as measured by fetal liver H₂O₂ and MDA.     

Gestational changes in concentrations of selenium and zinc in the porcine fetus and the effects of maternal intake of selenium

The effect of maternal dietary selenium (Se) and gestation on the concentrations of Se and zinc (Zn) in the porcine fetus were determined. Mature gilts were randomly assigned to treatments of either adequate (0.39 ppm Se) or low (0.05 ppm Se) dietary Se. Gilts were bred and fetuses were collected throughout gestation. Concentrations of Se in maternal whole blood and liver decreased during gestation in sows fed the low-Se diet compared to sows fed the Se-supplemented diet. Maternal intake of Se did not affect the concentration of Se in the whole fetus; however, the concentration of Se in fetal liver was decreased in fetuses of sows fed the low-Se diet. Although fetal liver Se decreased in both treatments as gestation progressed, the decrease was greater in liver of fetuses from sows fed the low-Se diet. Dietary Se did not affect concentrations of Zn in maternal whole blood or liver or in the whole fetus and fetal liver. The concentration of Se in fetal liver was lower but the concentration of Zn was greater than in maternal liver when sows were fed the adequate Se diet. These results indicate that maternal intake of Se affects fetal liver Se and newborn piglets have lower liver Se concentrations compared to their dams, regardless of the Se intake of sows during gestation. Thus, the piglet is more susceptible Se deficiency than the sow.     

Dietary regulation of maternal and fetal cholesterol metabolism in the guinea pig

Studies to determine the effects of pre-natal interventions on maternal and fetal cholesterol homeostasis were carried out in the guinea pig. Guinea pig dams were fed either non-purified guinea pig diet or diet supplemented with either 1.1% of the bile acid binding resin cholestyramine or 0.25% cholesterol. Whole body rates of endogenous cholesterol synthesis were determined by quantitation of [3H]water incorporation into digitonin precipitable sterols in non-pregnant animals and at 40 and 60 days of gestation in the dam and fetus. Maternal hepatic cholesterol synthesis was reduced 87% by dietary cholesterol and was increased 3.5-fold with cholestyramine feeding. Fetal hepatic and peripheral tissue cholesterol synthesis rates peaked at 40 days gestation when peripheral tissue cholesterol synthesis was 5.7-fold higher and hepatic synthesis 6.2-fold greater than the near adult levels observed at 60 days. Cholesterol synthesis in the fetus was relatively insensitive to dietary manipulations; however, maternal cholestyramine treatment did result in a 1.4-fold increase in fetal carcass cholesterol synthesis at 60 days gestation. These data demonstrate that maternal cholesterogenic systems maintain responsiveness to dietary regulation during pregnancy; whereas fetal cholesterol homeostasis is relatively insensitive to dietary cholesterol throughout gestation yet may respond to induction by maternal cholestyramine treatment during the late gestation period.     

Antenatal antioxidant prevents adult hypertension, vascular dysfunction, and microvascular rarefaction associated with in utero exposure to a low-protein diet

Developmental programming of hypertension is associated with vascular dysfunction characterized by impaired vasodilatation to nitric oxide, exaggerated vasoconstriction to ANG II, and microvascular rarefaction appearing in the neonatal period. Hypertensive adults have indices of increased oxidative stress, and newborns that were nutrient depleted during fetal life have decreased antioxidant defenses and increased susceptibility to oxidant injury. To test the hypothesis that oxidative stress participates in early life programming of hypertension, vascular dysfunction, and microvascular rarefaction associated with maternal protein deprivation, pregnant rats were fed a normal, low protein (LP), or LP plus lazaroid (lipid peroxidation inhibitor) isocaloric diet from the day of conception until delivery. Lazaroid administered along with the LP diet prevented blood pressure elevation, enhanced vasomotor response to ANG II, impaired vasodilatation to sodium nitroprusside, and microvascular rarefaction in adult offspring. Liver total glutathione was significantly decreased in LP fetuses, and kidney eight-isoprostaglandin F2alpha (8-isoPGF(2alpha)) levels were significantly increased in adult LP offspring; these modifications were prevented by lazaroid. Renal nitrotyrosine abundance and blood levels of 1,4-dihydroxynonene and 4-hydroxynonenal-protein adducts were not modified by antenatal diet exposure. This study shows in adult offspring of LP-fed dams prevention of hypertension, vascular dysfunction, microvascular rarefaction, and of an increase in indices of oxidative stress by the administration of lazaroid during gestation. Lazaroid also prevented the decrease in antioxidant glutathione levels in fetuses, suggesting an antenatal mild oxidative stress in offspring of LP-fed dams. These studies support the concept that perinatal oxidative insult can lead to permanent alterations in the cardiovascular system development.     

Maternal diet and dioxin-like activity, bulky DNA adducts and micronuclei in mother-newborns

Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006-2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet was estimated on the basis of maternal food frequency questionnaire (FFQ) completed by the end of pregnancy. Biomarkers were detected in paired blood samples through the dioxin-responsive chemical-activated luciferase expression (CALUX)(?) bioassay, (32)P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal (β 95%CI; 0.46 (0.08, 0.84)) and cord blood (β 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size.     

The effects of hydrocortisone on lung structure in fetal lambs

The effect of cortisol infusion on fetal lung development was studied in lambs. Changes were compared with those of control groups of saline-infused fetuses of the same age (day 132) and normal late gestation fetuses (142 +/- 4.6 days). Cortisol was infused into five fetal lambs at 129 days of gestation at a rate of 17.0 mg/day. Four fetuses were delivered by hysterotomy at the onset of labour-like uterine activity (58 +/- 3 h). In cortisol-infused fetuses the concentration of cortisol in fetal plasma and tracheal fluid rose to levels similar to those in normal fetuses during the last week of gestation. Progesterone concentration in maternal plasma declined at about 48 h after the start of treatment. Cortisol-infused lambs showed increases in fixed lung volume, specific lung volume, absolute volume of both parenchyma and non-parenchyma and the proportion of the parenchyma which was potential airspace and a decrease in the proportion of parenchyma. For cortisol-infused lambs Type II cell size and the abundance of lamellar bodies, and the volume fraction of cell occupied by the nucleus were similar to the 142 day group, whereas Golgi apparatus and RER were closer to age matched saline-infused (day 132) controls. Glycogen content was midway between the two control groups. We conclude that infusion of cortisol for about 60 h at physiological levels, beginning at 0.85 of gestation, accelerates many, but not all aspects of pulmonary parenchymal maturation, expressed in terms either of morphogenesis of the gas exchange area or differentiation of Type II alveolar cells.     

Role of feline maternal taurine nutrition in fetal cerebellar development: An immunohistochemical study

Summary We report the effects of four levels of maternal dietary taurine on the cerebellum of 45-day gestation fetuses. As we have previously reported for newborn and 8-week-old kittens, maternal dietary taurine content has a profound effect also on fetal cerebellum. Fetuses from queens fed the lowest amount of taurine had the greatest density of granule cells, probably because of smallest brain size, and had a high proportion of morphological abnormalities. Somewhat surprising was the observation that the fetuses from the lowest maternal dietary taurine group had the highest proportion of taurine-positive granule cells. In addition, these results confirm the vulnerability of developing fetal brain to its intrauterine environment.     

Formation of benzo[a]pyrene-DNA adducts by microsomal enzymes: comparison of maternal and fetal liver, fetal hematopoietic cells and placenta

The formation of benzo[a]pyrene (BP)-DNA adducts was studied in vitro in the presence of microsomes prepared from the isolated labyrinth zone of the rat placenta, the hematopoietic erythroblast cells of the fetal liver, the fetal liver, as well as the maternal liver. Pregnant rats received beta-naphthoflavone (beta NF; 15 mg/kg, i.p.) on day 17 gestation. One day later, placentae, fetal and maternal livers were obtained and hematopoietic erythroblast cells were separated from hepatocytes in the fetal livers. The respective microsomal fractions were incubated in the presence of calf thymus DNA, NADPH-regenerating system and [3H]BP (300 microCi) at 37 degrees C for 30 min. Following beta NF pretreatment, the levels of covalent binding (pmol/mg DNA/mg microsomal protein) for maternal liver, fetal liver, placenta and erythroblast cells were: 28.4, 2.4, 0.31 and 3.9, respectively, with the hematopoietic erythroblast cells being the most active among fetal tissue preparations. The extent of transplacental induction compared to control was greatest in the hematopoietic cells (18-fold) followed by fetal liver (16-fold) and labyrinth zone (5-fold). Further experiments characterized the BP-DNA adducts formed by induced microsomes. DNA was isolated, purified and digested sequentially with DNase I, snake venom phosphodiesterase type II and alkaline phosphatase type III. The deoxynucleoside-BP adducts were purified on a Sephadex LH-20 column and then separated on HPLC and the adducts were quantitated radiometrically. Seven distinct adducts were separated on HPLC and named A-G in order of elution. Adduct B was prominent in all preparations (22-55% total radioactivity). The adduct profile and retention time for peak B is similar to that reported for the adduct formed by microsomal activation of 9-hydroxy BP. Peak D constituted a major fraction (19%) in maternal liver profiles in comparison with the three fetal tissue preparations (8%). In subsequent experiments, peak D was shown to be derived from reaction of (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) with DNA. Peak C was unique to erythroblast cell and labyrinth profiles, while peak G was specific for maternal liver and fetal liver profiles. These results demonstrate that fetal liver and its hematopoietic cells are significant sites of BP bioactivation which may contribute to the fetal toxicity of polyaromatic hydrocarbons.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.