Messenger RNA for glutamine synthetase: its partial purification, translation in a cell-free system and its regulation by hydrocortisone.

We have verified that the enzymatic hydroxylation of carcinogenic aromatic hydrocarbons produces product molecules in electronically excited states. Introduction of the carcinogen benzo[α]pyrene into liver microsomes from 3-methylcholanthrene-induced rats results in a significant chemiluminescence which is shown for the first time to be correlated with the enzymatic hydroxylation of the parent carcinogen. The kinetics of the chemiluminescence implicate the intermediate epoxide as the precursor to the excited state product molecules.     

Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity.

In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.     

Relative roles of K region and bay region towards determining the carcinogenic potencies of polycyclic aromatic hydrocarbons

Using the quantum mechanical MINDO/3 method a bond reactivity index based on superdelocalization of electrons in a compound has been formulated in order to estimate the relative carcinogenic properties of polycyclic aromatic hydrocarbons. The values of the index calculated for the K region as well as the bay region suggest that a knowledge of the reactivities of both regions is necessary for a reasonable estimation of the relative carcinogenic potencies of PAHs. However, no direct relationship between the two reactivity indices was observed in the calculations. It was found that the results for the bay-region reactivity index correlated well with the relative carcinogenic potencies of the molecules.     

Polycyclic aromatic hydrocarbons as plausible prebiotic membrane components

Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C(6)-C(10) fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.     

The formation and occurrence of polynuclear aromatic hydrocarbons associated with food

Polynuclear aromatic hydrocarbons are common contaminants of processed food, usually at trace levels. These hydrocarbons are products of combustion and pyrolysis, and are present in petroleum and coal, and in products derived from them. Most polynuclear aromatic hydrocarbons are not carcinogenic, but some of them are, and a few are potent inducers of skin and lung tumors in mice. Their carcinogenic properties have not been fully explored, but they seem to be less potent by ingestion or inhalation, and they are known as a group to produce cancer in humans. The most effective carcinogens among them are those with 5 or 6 fused rings, and these tend to be less prevalent in mixtures than the 3- and 4-ring hydrocarbons, most of which are not carcinogenic. Sophisticated analytical methods, using solvent extraction and chromatography have been developed to detect and measure polynuclear aromatic hydrocarbons at levels of 1 in 10(9) (1 part per billion) or less, and these have been applied to the measurement of individual compounds in foods, as well as in products of combustion and pyrolysis. Wood smoke and smoked foods contain the carcinogenic benzo[a]pyrene at levels of 1 ppb, and other hydrocarbons; liquid smoke has lower levels. Crude vegetable oils have higher concentrations, but purified 'deodorized' oils have benzo[a]pyrene levels near 1 ppb. Sausages cooked over burning logs had as much as 200 ppb benzo[a]pyrene. Charcoal-broiled steaks and ground meat had benzo[a]pyrene concentrations up to 50 micrograms/kg, while less fatty pork and chicken had lower concentrations (up to 10 micrograms/kg). It was probable that the rendered fat dripped on to the hot charcoal and pyrolyzed to form quantities of polynuclear aromatic hydrocarbons, which rose with the smoke to deposit on the meat. Therefore, oven cooking or cooking with a heat source above the meat, or segregation of the meat from the smoke resulted in food containing negligible amounts of polynuclear aromatic hydrocarbons. Modifications of cookings practices accordingly would greatly reduce exposure to this group of carcinogens.     

Measurement of DNA-excision repair in suspensions of freshly isolated rat hepatocytes after exposure to some carcinogenic compounds: its possible use in carcinogenicity screening.

When suspensions of freshly isolated rat hepatocytes were exposed to a number of carcinogenic compounds, it was possible to measure an increased UDS by a rapid procedure via liquid-scintillation counting. For a number of carcinogenic compounds and some of their non-carcinogenic structural analogues a good correlation between the carcinogenic property and the ability to induce UDS was demonstrable. Out of 12 carcinogenic compounds, belonging to several different chemical classes, 10 gave rise to an increased UDS, whereas only 2 compounds, the polycyclic aromatic hydrocarbons benzo[alpha]pyrene and benz[alpha]anthracene, did not. All 4 noncarcinogenic compounds tested were negative. Possibly this method can be of value as a routine screening test, in combination with other short-term test systems, thus improving the predictive value of screening in vitro with respect to carcinogenicity.     

A study on the perturbation of model lipid membranes by phenoxazines

The interactions of six newly synthesized phenoxazine derivatives with lipid bilayers were studied by means of calorimetry, fluorescence spectroscopic methods and electron spin resonance. Depending on their structure studied compounds decreased membrane fluidity and increased lipid order in liquid-crystalline bilayers to different degrees. These studies showed also that phenoxazine molecules are located close to the polar/apolar interface of bilayer. The results allow to conclude that phenoxazines rather weakly interact with lipid bilayers.     

Interaction of cytochrome P-450 with hydrocarbons

Hydrocarbons of different structures interact with microsomal and solubilized cytochrome P-450 from liver of phenobarbital-pretreated rats forming a high spin enzyme-substrate type complex. The affinity of cytochrome P-450 for hydrocarbons increases with increasing lipophilicity independently of the chemical structure. The binding capacity of microsomal P-450 for aliphatic hydrocarbons is generally higher than for aromates. Mutual influence in binding of two different hydrocarbons by microsomal P-450 is stronger among aromatic than among aliphatic hydrocarbons; in both cases it appears to be effected rather by specific interaction of both substances with the binding site than by a nonspecific influence on the microsomal membrane. Only one fraction of low spin form of solubilized cytochrome P-450 from rat liver interacts with hydrocarbons. The binding capacity for aromatic and aliphatic substances corresponds to that found in microsomes. The affinity for the most lipiphilic substrate, perhydrophenanthrene, is equal in microsomal and solubilized preparation; with decreasing lipophilicity the affinity of solubilized P-450 decreases faster than in microsomes. The LM2 fraction of cytochrome P-450 from phenobarbital-pretreated rabbits interacts only with aliphatic hydrocarbons with wide variation of the binding capacity. The affinity is generally one order of magnitude lower than in microsomes. Active fractions of solubilized P-450 from both species are rapidly converted to P-420 by dithionite. The extent of this conversion is strongly reduced by saturation with substrate.     

Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author's transl)

The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed.     

Behavior of spin labels in a variety of interdigitated lipid bilayers

The behavior of a number of spin labels in several lipid bilayers, shown by X-ray diffraction to be interdigitated, has been compared in order to evaluate the ability of the spin label technique to detect and diagnose the structure of lipid bilayers. The main difference between interdigitated and non-interdigitated gel phase bilayers which can be exploited for determination of their structure using spin labels, is that the former have a much less steep fluidity gradient. Thus long chain spin labels with the nitroxide group near the terminal methyl of the chain, such as 16-doxylstearic acid, its methyl ester, or a phosphatidylglycerol spin label containing 16-doxylstearic acid (PG-SL), are more motionally restricted and/or ordered in the interdigitated bilayer than in the non-interdigitated bilayer. This difference is large enough to be of diagnostic value for all three spin labels in the interdigitated bilayers of dihexadecylphosphatidylcholine, dipalmitoylphosphatidylcholine/ethanol, and 1,3-dipalmitoylphosphatidylcholine. However, it is not large enough to be of diagnostic value at low temperatures. Use of probes with the nitroxide group closer to the apolar/polar interface reveals that these latter interdigitated bilayers are more disordered or less closely packed. As the temperature is increased, however, the motion of the PG-SL does not increase as much in these interdigitated bilayers as in non-interdigitated bilayers. The difference in the motion and/or order of PG-SL between interdigitated and non-interdigitated bilayers is large enough at higher temperatures to be of value in diagnosing the structure of the bilayers. Thus by choice of a suitable spin label and a suitable temperature, this technique should prove useful for detection and diagnosis of lipid bilayer structure with a good degree of reliability. Caution must, of course be exercised, as with any spectroscopic technique. Spin labels will also be invaluable for more detailed studies of known interdigitated bilayers, which would be time- and material-consuming, if carried out using X-ray diffraction solely.     

Ah receptor-independent inhibition of gap junction intercellular communications in hepatoma 27 cell culture by polycyclic aromatic hydrocarbons

Gap junction intercellular communication (GJIC) is a system that regulates tissue homeostasis. It is generally accepted that GJIC down-regulation is linked to carcinogen tumor-promoting properties. The effect of carcinogenic polycyclic aromatic hydrocarbons on GJIC in cultured hepatoma 27 cells deficient in cytochrome P450 and Ah receptor has been investigated. It was found that, out of six compounds examined, only benz/a/pyrene and 3-methylcholanthrene were able to inhibit GJIC. It is concluded that in hepatoma cells there is unknown factor that interacts with some polycyclic aromatic hydrocarbons and inhibit GJIC. Application of benz/a/pyrene together with benz/e/pyrene (an agent with similar structure but without carcinogenic activity) showed that GJIC inhibition by benz/a/pyrene is at least a two-stage process.     

ESR spectral analysis of the molecular motion of spin labels in lipid bilayers and membranes based on a model in terms of two angular motional parameters and rotational correlation times.

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide spin-labeled molecule undergoing rapid restricted rotations (twisting) about its long molecular axis while simultaneously tumbling within a cone. Explicit expressions are derived for the hyperfine splittings and g-values, as well as for the secular contributions to the motionally modulated linewidths. The present model is useful for analyzing the restricted twisting and tumbling motions, and rotational correlation times, of spin-labeled molecules in bilayers. Simulated spectra compare well with experimental spectra of lecithin bilayers marked with cholestane spin label, over a wide temperature range.     

The vibrational spectrum of water in liquid alkanes.

The water wire hypothesis of hydrogen-ion transport in lipid bilayers has prompted a search for water aggregates in bulk hydrocarbons. The asymmetric stretching vibration of the water dissolved in n-decane and in a number of other alkanes and alkenes has been observed. The water band in the alkanes is very wide and fits to the results of a J-diffusion calculation for the water rotation. This implies that the water is freely rotating between collisions with the solvent and certainly not hydrogen bonded to anything. The existence of water aggregates is thus most unlikely. In contrast, water in an alkene is hydrogen bonded to the solvent molecules (although not to other water molecules) and shows an entirely different spectrum.     

Direct electron spin resonance evidence for alpha-tocopherol-induced phase separation in model membranes

The high resolution (narrow linewidth) amphiphilic spin probe perdeutero di-t-butyl nitroxide (PDDTBN) has been used to investigate the effect of alpha-tocopherol on lecithin liposomes. The electron spin resonance (ESR) results obtained as a function both of alpha-tocopherol concentration and of temperature indicate the presence of two different hydrophobic sites for the spin probe molecules. The presence of two distinct phases, one alpha-tocopherol-poor and the other alpha-tocopherol-rich, is suggested in these phospholipid bilayers.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Inhibition of Gap Junction Intercellular Communications in Cell Culture by Polycyclic Aromatic Hydrocarbons (PAH) in the Absence of PAH Metabolism

We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.     

Motional restrictions of membrane proteins: a site-directed spin labeling study

Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch.     

Roles of UDP-glucuronosyltransferases in chemical carcinogenesis

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the biliary or urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme superfamily that control the glucuronidation of metabolites of aromatic hydrocarbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be coinduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the "toxin-resistance phenotype" at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.     

Mammalian cell transformation and cell-mediated mutagenesis by carcinogenic polycyclic hydrocarbons.

The introduction of a polycyclic hydrocarbon such as benzo(alpha)pyrene (BP) into normal golden hamster embryo cell cultures results, in addition to cytotoxicity, in malignant cell transformation. Studies on the effect of different doses of BP on the normal cells showed that the frequency of transformed colonies was directly related to the dose of the carcinogen. Analysis of this dose-response curve suggests a one-event ("one-hit") response for transformation by this carcinogen. The one-event response for transformation by carcinogenic polycyclic hydrocarbons and the fact that these carcinogens bind to DNA in susceptible cells suggests that transformation can involve a single alteration in the genetic constitution of the treated cells. Carcinogens may, therefore, produce somatic mutations, some of which may involve the genes that control malignancy. Recently, considerable progress has been made in developing models for the study of chemical mutagenesis in mammalian cells. Using resistance to 8-azaguanine as a marker, positive correlations between mutagenicity and transformation were obtained with chemically reactive carcinogens such as N-acetoxy-N-2-fluorenyl-acetamide, N-methyl-N'-nitro-N-nitrosoguanidine and K-region epoxides of polycyclic hydrocarbons. However, no such correlations were obtained with the carcinogenic polycyclic hydrocarbons themselves, since the cell lines used in chemical mutagenesis do not metabolize these carcinogens. In order to obtain better correlations, we have developed a cell-mediated mutagenic assay with carcinogenic hydrocarbons in which Chinese hamster cells, which are susceptible for mutagenesis, were co-cultivated with lethally irradiated rodent cells that can metabolize these compounds. Using this cell mediated assay, we obtained mutagenesis with the carcinogenic hydrocarbons 7,12-dimethylbenz(alpha)anthracene (DMBA), BP, 3-methylcholanthrene and 7-methylbenz(alpha)anthracene; the most potent carcinogen, DMBA, gave the highest frequency of mutations. The polycyclic hydrocarbons, pyrene and benz(alpha)anthracene, which are not carcinogenic were also not mutagenic. We have therefore demonstrated a relationship between the carcinogenecity of polycyclic hydrocarbons and their mutagenicity in mammalian cells, without having to isolate their reative metabolic intermediates. It should be possible to use in this system human cells from different organs and individuals to screen for environmental chemicals hazardous to humans which have to be metabolically activated.     

Induction of aldehyde dehydrogenase by polycyclic aromatic hydrocarbons in rats

Short-term intragastric administration of selected polycyclic aromatic hydrocarbons (100 mg/kg daily for 4 days) to male Wistar rats resulted in marked changes in liver cytosolic aldehyde dehydrogenase activity. Non-carcinogenic anthracene, phenanthrene and chrysene produced a 2.5–3-fold increase in the activity assayed with propionaldehyde as substrate and NAD as coenzyme. Weakly carcinogenic 1,2-benzanthracene enhanced aldehyde dehydrogenase activity 9-fold and the potent carcinogens 3,4-benzpyrene and 3-methylcholanthrene 30-fold. With benzaldehyde as substrate and NADP as coenzyme the differences between the groups were even more pronounced. Somewhat similar but less manifest effects on aldehyde dehydrogenase activity were detected also in the liver microsomes and in the postmitochondrial fractions of the small intestinal mucosa. On the basis of their ability to induce aldehyde dehydrogenase activity the compounds could be divided into three groups. This classification was found to correlate well with the carcinogenic potency of the compounds. It appeared that the exposure to polycyclic aromatic hydrocarbons, especially the carcinogenic ones, was followed by synthesis of a new aldehyde dehydrogenase form. This new form was differentiated from the normally existing cytosolic aldehyde dehydrogenase by its ability to oxidize benzaldehyde in the presence of NADP.