Werner Lutz

Essential function of p300 acetyltransferase activity in heart, lung and small intestine formation

p300 and CBP are large nuclear acetyltransferases exhibiting a complex multi‐domain structure. Mouse embryos nullizygous for either p300 or Cbp die at midgestation, while heterozygotes are viable but in part display defects in neurulation or bone morphogenesis. To directly examine the contribution of the acetyltransferase (AT) activity to mouse development, we have abrogated this function by a knock‐in approach. Remarkably, a single AT‐deficient allele of p300 or Cbp leads to embryonic or neonatal lethality, indicating that the mutant alleles are dominant. Formation of the cardiovascular system, the lung and the small intestine are strongly impaired in p300 AT and to a much lesser extent in Cbp AT mutant embryos, a difference that is also reflected by the defects in gene expression. Embryonic stem cells homozygous for either the p300 AT or a p300 null mutation respond differently to BMP2 stimulation, indicating that the two alleles are not equivalent. Unexpectedly, the p300 AT‐mutant cells upregulate BMP‐inducible genes to levels similar or even higher than observed in wild‐type cells.

Differential role of p300 and CBP acetyltransferase during myogenesis: p300 acts upstream of MyoD and Myf5

Studies in tissue culture cells have implicated p300 and CBP acetyltransferases in myogenic regulatory factor (MRF) mediated transcription and terminal differentiation of skeletal muscle cells. However, in vivo data placing p300 and CBP on myogenic differentiation pathways are not yet available. In this report we provide genetic evidence that p300 but not CBP acetyltransferase (AT) activity is required for myogenesis in the mouse and in embryonic stem (ES) cells. A fraction of embryos carrying a single p300 AT‐ deficient allele exhibit impaired MRF expression, delayed terminal differentiation and a reduced muscle mass. In mouse embryos lacking p300 protein, Myf‐5 induction is severely attenuated. Similarly, ES cells homozygous for a p300 AT or a p300 null mutation fail to activate Myf5 and MyoD transcription efficiently, while Pax3, acting genetically upstream of these MRFs, is expressed. In contrast, ES cells lacking CBP AT activity express MyoD and Myf5 and undergo myogenic differentiation. These data reveal a specific requirement for p300 and its AT activity in the induction of MRF gene expression and myogenic cell fate determination in vivo.

Quantitative evaluation of DNA binding data for risk estimation and for classification of direct and indirect carcinogens

SummaryInvestigation of covalent DNA binding in vivo provided evidence for whether a test substance can be activated to metabolites able to reach and react with DNA in an intact organism. For a comparison of DNA binding potencies of various compounds tested under different conditions, a normalization of the DNA lesion with respect to the dose is useful. A covalent binding index, CBI=(μmol chemical bound per mol DNA nucleotide)/(mmol chemical administered per kg body weight) can be determined for each compound. Whether covalent DNA binding results in tumor formation is dependent upon additional factors specific to the cell type. Thus far, all compounds which bind covalently to liver DNA in vivo have also proven to be carcinogenic in a long-term study, although the liver was not necessarily the target organ for tumor growth. With appropriate techniques, DNA binding can be determined in a dose range which may be many orders of magnitude below the dose levels required for significant tumor induction in a long-term bioassay. Rat liver DNA binding was proportional to the dose of aflatoxin B1 after oral administration of a dose between 100 μg/kg and 1 ng/kg. The lowest dose was in the range of general human daily exposures. Demonstration of a lack of liver DNA binding (CBI<0.1) in vivo for a carcinogenic, nonmutagenic compound is a strong indication for an indirect mechanism of carcinogenic action. Carcinogens of this class do not directly produce a change in gene structure or function but disturb a critical biochemical control mechanism, such as protection from oxygen radicals, control of cell division, etc. Ultimately, genetic changes are produced indirectly or accumulate from endogenous genotoxic agents. The question of why compounds which act via indirect mechanisms are more likely to exhibit a nonlinear range in the dose-response curve as opposed to the directly genotoxic agents or processes is discussed.

MycN and IFNγ cooperate in apoptosis of human neuroblastoma cells

Neuroblastomas undergo spontaneous regression at an unusually high rate. The mechanisms are not clear, but apoptosis may be involved. A large proportion of neuroblastomas is characterized by amplification of MYCN. Using human neuroblastoma cells harbouring tetracycline controlled expression of MYCN we have analysed the role of the MycN protein and IFNγ in cell death decision. Neither conditional expression of MYCN nor treatment with IFNγ alone was sufficient to trigger cell death. However, when acting in concert MycN and IFNγ efficiently triggered cell death, which was accompanied by DNA fragmentation and required caspase activity, two hallmarks of apoptosis. MycN and IFNγ may cooperate along at least two different pathways. First, IFNγ increased the CD95 cell surface expression while MycN enhanced the cellular susceptibility for the CD95 mediated death signal. Second, IFNγ treatment induced expression of BAK mRNA while MycN and IFNγ in combination increased the amount of Bax protein, another activator of apoptosis, without a concomitant increase in BAX mRNA. MycN also increased cell death in response to TRAIL and TNFα, suggesting that enforced MYCN expression in general increases the susceptibility of neuroblastoma cells towards a variety of death stimuli.

Assessment of the risk of formation of carcinogenic N-nitroso compounds from dietary precursors in the stomach.

A literature review has shown that the daily intakes of various N-nitroso-precursor classes in a typical European diet span five orders of magnitude. Amides in the form of protein, and guanidines in the form of creatine and creatinine, are the nitrosatable groups found most abundantly in the diet, approaching levels of 100 g/day and 1 g/day, respectively. Approximately 100 mg of primary amines and amino acids are consumed daily, whereas aryl amines, secondary amines and ureas appear to lie in the 1-10 mg range. The ease of nitrosation of each precursor was estimated, the reactivities being found to span seven orders of magnitude, with ureas at the top and amines at the bottom of the scale. From this information and an assessment of the carcinogenicity of the resulting N-nitroso derivatives, the potential health risk due to gastric in vivo nitrosation was calculated. The combined effects of these risk variables were analysed using a simple mathematical model: Risk = [daily intake of precursor] X [gastric concentration of nitrite]n X [nitrosatability rate constant] X [carcinogenicity of derivative]. The risk estimates for the various dietary components spanned nine orders of magnitude. Dietary ureas and aromatic amines combined with a high nitrite burden could pose as great a risk as the intake of preformed dimethylnitrosamine in the diet. In contrast, the risk posed by the in vivo nitrosation of primary and secondary amines is probably negligibly small. The risk contribution by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes. Thus three priorities for future work are a comprehensive study of the sources and levels of arylamines and ureas in the diet, determination of the carcinogenic potencies of key nitrosated products to replace the necessarily vague categories used so far, and the development of short-term in situ tests for studying the alkylating power of genotoxicity of N-nitroso compounds too unstable for inclusion in long-term studies.

Mechanism of the carcinogenic action of benzene: irreversible binding to rat liver DNA.

Introduction It has been established that exposure of animals and men to benzene (see Ref. 1 for a recent review on benzene toxicity) can result in darnage to the haematopoietic system and a relationship between such exposure and the development of leukemia is suggested by many case reports [2]. Since a common feature of chemical carcinogens is their interaction with biological macromolecules [ 3] , most probably a covalent binding of a reactive metabolite to DN A, and since the carcinogenicity of benzene to animals has not yet been unequivocally proved, we thought it necessary to examine the interaction of benzene with DNA. We have exposed adult male rats to radioactive benzene (tritium or carbon-14 label) in a closed inhalation chamber and were able to show for the first time a covalent binding of a benzene metabolite to DN A in vivo. The liver was chosen as a model system since it is the main organ for the metabolism of foreign compounds containing all the enzymes necessary for an activation and since this organ provides ample DN A for the determination of low specific radioactivity.

SACCHARIN DOES NOT BIND TO DNA OF LIVER OR BLADDER IN THE RAT

Introduction Saccharin was found to induce bladder tumours in male rats of the first and the second generation after life-long feeding of a diet containing 5% saccharin [1]. On the other band, with lower doses, and in epidemiological sturlies on the human consumption as sweetening agent there is no clear evidence for a carcinogenic activity despite the long and widespread use [ 2] . This controversy prompted us to investigate the interaction of saccharin with DNA in an intact mammalian organism. Most of the known chemical carcinogens undergo a covalent binding to DNA [3], and the resulting darnage can lead to a mutation. Saccharin has not been found to be mutagenic [ 4,5] and the present study shows that it does not undergo a covalent binding to DNA of the liver or the bladder of male rats either. The tumor induction is therefore probably due to a secondary darnage to the bladder.

Investigation of the potential for binding of di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo

It was the aim of this investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or without pretreatment for 4 weeks with 1% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [14C]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The limit of detection, CBI less than 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [14C]- and [3H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside or base adducts are expected. The respective limits of detection were at 0.07 and 0.04 CBI units for the 14C and 3H labels, respectively. The experiments with [14C]- and [3H]DEHA, labeled in the alcohol moiety and administered to mice, revealed a minute radioactivity of less than 50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which had been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for 14C and 0.01 to 0.12 for 3H resulted. Determination of the level of 14CO2 expiration revealed a linear correlation with the specific activity of DNA. Experiments with 2-ethyl[1-14C]hexanol performed with both rats and mice allowed the conclusion that most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01.(ABSTRACT TRUNCATED AT 400 WORDS)

Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes by phenobarbital, 3-methylcholanthrene, 2,3,7,8-tetrachloro-dibenzo-p-dioxin, dieldrin and other inducers.

Abstract Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat liver nuclei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo- p -dioxin (TCDD, 0.01 mg/kg) with an induction to 500 and 300 per cent for nuclei and microsomes, respectively, after 2 days, and to 400 per cent for both after 12 days. Phenobarbital (PB) was given continuously in the drinking water (1 g/l) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. the nuclear activity was only slightly induced to a Constant level of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daily i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down to control values after 12 days. Other inducers tested were benz[a]anthracene (BA), hexachlorobenzene (HCB) and 1,1,1-trichloro-2,2- bis ( p -chlorophenyl)ethane (DDT). The induction pattern with BA was similar to that of MC, a model compound for the group of cytochrome P448 inducers. the induction by HCB and DDT resembled that by PB, a typical cytochrome P450 inducer.

Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene

2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue mice). The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10(5) plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10(-5) (pooled results of two animals, 8 mutant plaques/289,530 plaques). At 300 ppm in the diet, the rate of 3.5 x 10(-5) (8/236,300) was not significantly increased over background. At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 x 10(-5) (17/221,240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF.