Critical parameters for adduct formation of the carcinogen (+)-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide with oligonucleotides.

Various parameters relevant for the formation of dG adducts produced in the reaction of individual benzo[a]pyrene diol epoxide (BPDE) stereoisomers with oligonucleotides have been studied. Reaction time, temperature, pH, molar ratio of diol epoxide and oligonucleotide, base sequence, and buffer system were shown to affect the amount of (+)-anti-BPDE dG adducts formed. Optimum experimental conditions for dG adduct formation were different depending on the base sequence context of the oligonucleotide employed [5'-d(CCTATAGATATCC) or 5'-d(CCTATTGCTATCC)]. In general, low temperature to allow a longer reaction time, slightly alkaline Tris-HCl (pH 7.5-8.0) or alkaline phosphate buffer (pH 11), l…

Stereoselective Metabolic Activation of Dibenzo[a,l]Pyrene in the Human Mammary Carcinoma Cell Line MCF-7 Results in Formation of (-)-antiand (+)-syn-11,12-Diol-13,14-Epoxidedeoxyadenosine Adducts in DNA

Abstract Dibenzo[a,l]pyrene (DB[a,l]P) is an important polycyclic aromatic hydrocarbon because of possible human exposure and its exceptionally high carcinogenicity in rodents. We examined the metabolism of DB[a,l]P and the formation of DB[a,l]P-DNA adducts in the human mammary carcinoma cell line (MCF-7). Analysis of the DNA adducts by 33P-postlabeling, immobilized boronate chromatography, HPLC and TLC demonstrated that DB[a,l]P is stereoselectively metabolized to specific optical isomers of DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE). The major anti-DB[a,l]PDE adduct formed in DB[a,l]P-treated MCF-7 cells resulted from reaction of (-)-anti-DB[a,l]PDE with DNA whereas the two major syn-…

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamste…

Abstract of the 68th Meeting (Spring Meeting) 6–9 March 1990, Heidelberg

Genetically engineered V79 Chinese hamster cells metabolically activate the cytostatic drugs cyclophosphamide and ifosfamide.

V79 cells, genetically engineered to express active cytochromes P450IIB1 and P450IA1, were used to study the cytotoxicity and mutagenicity of cyclophosphamide and ifosfamide. Cyclophosphamide, tested up to a concentration of 2 mM, was not cytotoxic in V79 nor in the P450IA1-expressing V79-derived cell line XEM2. Pronounced cytotoxicity was, however, observed in the P450IIB1-expressing V79-derived cell line SD1. Induction of gene mutations (acquisition of 6-thioguanine resistance) was observed in SD1 cells as well, but the effects were weak. Ifosfamide was inactive in V79 cells, but was cytotoxic in SD1 cells. Ifosfamide mustard, an active metabolite of ifosfamide, was equally cytotoxic and …

The response to isoproterenol of synaptic ribbon numbers in the rat pineal gland changes during postnatal development

Abstract In the mammalian pineal gland synaptic ribbons (SRs) are dynamic organelles of pinealocytes undergoing a day/night rhythm, with small numbers during daytime and significantly higher numbers at night, similar to the formation of the pineal hormone melatonin. Whereas the day/night rhythm of melatonin synthesis is adrenergically regulated, data on the adrenergic regulation of SR numbers in the rat pineal gland are at variance. While some authors have demonstrated that isoproterenol (ISO) stimulates SR numbers, others could not find any effect. To clarify the issue, we carried out identical experiments in two age groups. It was found that in male Sprague-Dawley rats, administration of …

Effects of LHRH, progesterone, estradiol-17 ? and dexamethasone in vitro on pineal synaptic ribbons and serotonin N-acetyltransferase activity in diestrous rats

Pineal glands of regularly cycling Sprague Dawley rats (180-220 g) killed on the diestrous morning (between 0900-1000 h) were incubated in appropriate media for six hours with LHRH (8.5 microM), progesterone (3.2 microM), estradiol-17 beta (370 nM) or dexamethasone (250 nM). Pineals incubated in hormone-free medium and unincubated glands served as controls. Six rats were used in each group. After incubation the glands were divided into two parts. One part was used to estimate serotonin N-acetyltransferase (NAT) activity. The other part was processed for electron microscopy to quantify synaptic ribbons (SR). The SR numbers were computed to 20,000 microns 2 area of pineal tissue. The number a…

In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland

The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide …

Molecular studies on the toxifying effects by genetically engineered cytochromes P450.

After almost two decades, it is now evident that methodology based on molecular biology and gene technology has dramatically changed the way basic and applied toxicology is being performed. It star...

Effects of electrical stimulation of the superior cervical ganglia on the number of ?synaptic? ribbons and the activity of melatonin-forming enzymes in the rat pineal gland

Melatonin metabolism in the mammalian pineal gland is under the clear influence of sympathetic fibers originating in the superior cervical ganglia (SCG). Previous studies suggested that pineal "synaptic" ribbons (SR) as well are regulated by the gland's sympathetic innervation. To gain more insight into the mechanisms involved, we examined the effects of sympathetic stimulation on SR number and on the activity of melatonin forming enzymes, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). The SCG in adult male rats were stimulated electrically during daytime for either 15 or 120 min. Immediately following stimulation, the glands were removed and processed fo…

Involvement of cyclic guanosine monophosphosphate (cGMP) and cytosolic guanylate cyclase in the regulation of synaptic ribbon numbers in rat pineal gland

In the rat pineal gland N-acetyltransferase (NAT) activity and synaptic ribbon (SR) numbers display a circadian rhythm. It is well-known that NAT activity is regulated by adrenergic mechanisms involving cyclic adenosine monophosphate (cAMP) as a second messenger. However, the mechanism involved in the regulation of SR numbers has not been established so far. In the present in vitro study, we have investigated the effects of 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), a cyclic guanosine monophosphate (cGMP) analog, and stimulation of guanylate cyclase on SR numbers. Incubation with 8-bromo-cGMP increased SR numbers in a dose- and time-dependent manner. Further, stimulation of the …

Covalent DNA adducts formed by benzo[c]chrysene in mouse epidermis and by benzo[c]chrysene fjord-region diol epoxides reacted with DNA and polynucleotides.

The metabolic activation in mouse skin of benzo[c]chrysene (B[c]C), a weakly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar and crude oil, was investigated. Male Parkes mice were treated topically with 0.5 mumol of B[c]C, and DNA was isolated from the treated areas of skin at various times after treatment and analyzed by 32P-postlabeling. Seven adduct spots were detected, at a maximum level of 0.89 fmol of adducts/microgram of DNA. Four B[c]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 mumol of the optically pure putative proximate carcinogens (+)- and (-)-trans-benzo[c]chrysene-9,10-dihydrodiols [(+)- and (-)-B[c]C-diols] led to th…

The role of arginine-vasopressin for pineal melatonin synthesis in the rat: involvement of vasopressinergic receptors.

Abstract The endogenously synthesized nonapeptide arginine vasopressin (AVP) is thought to be involved in transduction of photic information to the pineal gland. The enhancement of circulating AVP leads to a suppression of the nocturnal melatonin surge the mechanisms of which are unknown so far. We therefore studied the effect of dDAVP, an AVP analog with antidiuretic but without vasopressor activity, on pineal melatonin synthesis in Sprague-Dawley and AVP-deficient Brattleboro rats. The nocturnal intra-arterial application of dDAVP mimicked the inhibitory effect of AVP on the activity of the rate-limiting enzyme for pineal melatonin synthesis, N-acetyltransferase (NAT), in both rat strains…

p-Chlorophenylalanine treatment depresses the number of synaptic ribbon profiles in the rat pineal gland, but does not abolish their day-night rhythm

It is largely unknown as to how the day/night rhythm of pineal synaptic ribbon number is regulated. Transcriptional events related to the nocturnal formation of new synaptic ribbons occur early in the morning, when pineal serotonin levels begin to increase. The present study was carried out in order to elucidate as to how altered serotonin levels affect the day/night changes in the number of synaptic ribbon profiles. To this end, male Sprague-Dawley rats received a single dose of p-chlorophenylalanine (pCPA, 300 mg/kg body weight, 72 hours before sacrifice), which depresses tryptophan hydroxylase activity and hence pineal serotonin levels. Control animals received saline injections. Experim…

Synaptic ribbons of the rat pineal gland: responses to in-vivo and in-vitro treatment with inhibitors of protein synthesis.

To elucidate the role of protein synthesis in the nocturnal increase of synaptic ribbons in the rat pineal gland, actinomycin-D, which inhibits transcription, and cycloheximide, an inhibitor of translation, were used. To assure that the drugs were effective and to relate morphological changes to pineal biosynthetic phenomena, the activity of N-acetyltransferase and levels of pineal indoleamine were measured. Results of in-vivo, short-term and long-term treatment with either drug suggest that transcription of proteins related to synaptic ribbon formation occurs during the first half of the light phase, whereas translation takes place during the first few hours of the dark phase. In contrast,…

Detection of Reactive Quinones in the Metabolism of Polycyclic Aromatic Hydrocarbons by the Formation of their Glutathione Conjugates

Abstract The biotransformation of polycyclic aromatic hydrocarbons to quinones by rat liver microsomes was investigated. The employment of an electrochemical detector allowed the specific detection of quinones separated by reverse phase HPLC with higher sensitivity as compared to UV detection. Microsomal incubations of benzo[a]pyrene (BP) resulted in the formation of 1,6-, 3,6- and 6,12-quinones, of naphthalene in the detection of naphthalene-1,4-quinone, whereas ortho-quinones could only be detected in trace amounts. Additional protein binding studies showed that only 9–22% of synthetic ortho-quinones could be recovered from microsomal incubations. In order to scavenge possible reactive qu…

A possible role for cyclic guanosine monophosphate in the rat pineal gland.

Abstract Adrenergic stimulation of pinealocytes induces an increase of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). However, for cGMP no biological effects have been demonstrated so far. Therefore we tested the effects of the analog 8-bromo-cGMP on synaptic ribbon numbers and on melatonin synthesis as reflected by N -acetyltransferase (NAT) activity in the rat pineal gland in vitro. Incubation for 6 h with 8-bromo-cGMP did not change the activity of serotonin NAT but in increased the number of synaptic ribbons. These results indicate that cGMP is involved as a second messenger in the regulation of synaptic ribbon numbers in the rat pineal gland.

Covalent DNA adducts formed in mouse epidermis by benzo(g)chrysene

The metabolic activation in mouse skin of benzo[g]chrysene (B[g]C), a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar, was investigated. Male Parkes mice were treated topically with 0.5 micromol B[g]C and DNA was isolated from the treated areas of skin at various times after treatment and analysed by 32P-post-labelling. Seven major adduct spots were detected, at a maximum level of 6.55 fmol adducts/microg DNA. Mouse skin treated with the PAH benzo[c]phenanthrene (B[c]Ph) gave a total of 0.24 fmol adducts/microg DNA. B[g]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 micromol of the optically pure putative proximate carcinog…

The pineal complex in Roman high avoidance and Roman low avoidance rats.

Previous studies have shown that the pineal gland of Roman high avoidance (RHA/Verh) rats is larger than that of Roman low avoidance rats (RLA/Verh). In the present study measurement of enzyme activities (serotonin-N-acetyl-transferase, hydroxyindole-O-methyltransferase) revealed that pineals of RHA/Verh rats are twice as active in melatonin production than pineals of RLA/Verh rats. Indoleamine content was also higher in RHA/Verh rats, whereas noradrenaline content was the same in both lines. When values were expressed per mg protein, these differences disappeared except for N-acetyl-serotonin and noradrenaline which were higher or lower in RHA/Verh rats, respectively. Both lines had higher…

The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SA…

Scientific Proceedings Second International Symposium on Cytostatic Drug Resistance

Dihydrodiol Dehydrogenase: Substrate Specificity, Inducibility and Tissue Distribution

The present study shows that: Dihydrodiol dehydrogenase activity is present in the 100,000 g supernatant fraction of extrahepatic tissues. Dihydrodiol dehydrogenase is able to oxidize the hydroxy group and to reduce the keto group of a number of xenobiotics including quinones derived from polycyclic aromatic hydrocarbons. Dihydrodiol dehydrogenase was not inducible by various substances including hormones, polycyclic aromatic hydrocarbons, substrates of the enzyme and potent inducers of monooxygenases, epoxide hydrolase and glutathione S-transferases. Only in the case of thyroxine was a weak induction with a high dose of the hormone observed.