On the inhibition of microsomal drug metabolism by polychlorinated biphenyls (PCB) and related phenolic compounds.

The in vitro metabolism of p-nitroanisole, aminopyrine, and aniline by rat liver microsomal monoxygenases were studied in the presence of different polychlorinated biphenyl (PCB) mixtures and some related hydroxybiphenyls. The tested PCB mixtures contained preferably dichloro- (di-CB), tetrachloro- (tetra-CB), or hexachlorobiphenyls (hexa-CB). All PCB were competitive inhibitors of only aminopyrine demethylation by normal microsomes (Ki 22-39 micron). In microsomes of PCB-pretreated rats the aminopyrine demethylation was inhibited noncompetitively by di-CB and hexa-CB whereas tetra-CB remained a competitive inhibitor (Ki 12 micron). Moreover, after PCB pretreatment all PCB were competitive inhibitors of p-nitroanisole demethylation. 2-OH-biphenyl and 4-OH-biphenyl caused competitive inhibition of aminopyrine demethylation and aniline hydroxylation but failed to inhibit p-nitroanisole metabolism by normal microsomes. Chlorinated 4-hydroxybiphenyls inhibited competitively the metabolism of both type I and type II substrates. However, after PCB pretreatment all phenolic compounds caused uncompetitive inhibition of aniline hydroxylation.     

Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity

Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley-Liss, Inc.     

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.     

Distribution and biochemical effects of pentylenetetrazol in brains of swine treated with a polychlorinated biphenyl

In order to detect an effect on the CNS following treatment with polychlorinated biphenyls (PCB), pentylenetetrazol (PTZ) was administered to two groups of miniature swine. One group was a control and the other had been treated for 30 days with Aroclor 1254 (PCB) at a dose of 25 μg/kg in ground laboratory chow. No statistical difference was seen between the amount of PTZ administered or the time required to convulse for either group of animals. The total amount of PTZ found in brains of PCB-treated swine was less than from brains of control swine. Less PTZ was found in medulla and cerebellum from PCB-treated animals. The amount of PTZ found in motor cortex and in caudate nucleus from brains of PCB-treated swine was equal to the amounts found in similar structures of control brains. A 2–3-fold increase in dihydroxyphenylalanine (DOPA) levels was detected in motor cortex and medulla from PCB-treated animals, although levels of norepinephrine (NE) and gamma- aminobutyric acid (GABA) were unchanged from controls. It is concluded that PCB treatment had no effect on altering convulsive threshold of PTZ in swine, although PCB treatment does decrease the amount of PTZ in swine brains. Non-specific alterations in brain amine levels were also seen in PCB-treated swine.     

Enhancement of hepatic microsomal drug oxidation and glucuronidation in rat by 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT)

A single dose of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (160 mg/kg i.p.) enhanced the monooxygenase step of drug biotransformation in rat liver. The O-demethylation of p-nitroanisole was especially increased, a peak in activity approximately 5-fold compared with controls being attained in 7 days. On the other hand, there was only a 2-fold increase in aryl hydrocarbon hydroxylase activity.DDT increased the cytochrome P-450 content of the liver, this increase coincided well with that in p-nitroanisole O-demethylation activity.The UDPglucuronosyltransferase activity of liver microsomes was not enhanced by DDT administration, unless the microsomes were pretreated to reveal latent activity prior to assay. After trypsin digestion of microsomes a maximum increase in activity of approximately 3-fold was observed as a result of DDT dosage. The canonic surfactant cetylpyridinium chloride was less active in revealing the latent UDP-glucuronosyltransferase activity, and two other membrane perturbants, the detergent digitonin and phospholipase A, were unable to show enhancement in UDPglucuronosyltransferase as a result of DDT dosage.     

Mutagenicity research and testing in Sweden

A survey is given of Swedish legislation for control of chemicals in the environment. Although no direct legal requirements for mutagenicity testing of chemicals exist at present in Sweden, such requirements can be enforced within the existing laws.Testing and research in chemical mutagenicity are especially performed at the Environmental Toxicology Unit of the Wallenberg laboratory, University of Stockholm. An outline is given of the organization of the unit, which is based on an interdisciplinary cooperation, among divisions of organic and analytical chemistry, cellular toxicology, and genetics. As examples of projects under joint investigation results on polychlorinated biphenyl (PCB) and on vinyl chloride are briefly described.     

Separation of pure polychlorinated biphenyl isomers into two types of inducers on the basis of induction of cytochrome P-450 or P-448.

A number of isomerically pure polychlorinated biphenyls (PCBs) were tested as inducers of hepatic drug-metabolizing enzymes in the rat. The chlorinated biphenyl isomers can be categorized into two distinct groups of inducers, while commercial PCB mixtures have characteristics of both groups. Biphenyls chlorinated symmetrically in both the meta and para positions (3,4,3′,4′- and 3,4,5,3′,4′,5′-) increase the formation of cytochrome P-448, the ratio of the 455 to 430 peaks of the ethyl isocyanide difference spectrum, and aryl hydrocarbon hydroxylase and glucuronyl transferase activities, but decrease aminopyrine N-demethylase activity. These isomers are also the most toxic, as measured by weight loss. Biphenyl isomers chlorinated in both the para and ortho positions induce the formation of cytochrome P-450 rather than P-448, regardless of the chlorination of the meta position. These isomers, which include 2,4,2′,4′-tetra- and 2,4,5,2′,4′,5′-, 2,3,4,2′,3′,4′- and 2,4,6,2′,4′,6′-hexachlorobiphenyls, increase cytochrome P-450 and N-demethylase activity, but produce only a slight increase in aryl hydrocarbon hydroxylase activity, and do not alter the peak of the CO-difference spectrum or the ratio of the 455/430 peaks of the ethyl isocyanide difference spectrum. Isomers which are chlorinated in only one ring, or are chlorinated in both rings but not in the para positions, have very little activity as inducers of liver enzymes. Of the dichlorobiphenyls tested, 3,3′- and 4,4′-dichlorobiphenyls have very slight activity at extremely high doses.     

Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE_LB400) are largely unknown. BphAE_p4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE_LB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE_LB400 and BphAE_p4 and examine the biochemical properties of two BphAE_LB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.     

Induction of glutathione-S-transferase in the Northern quahog Mercenaria mercenaria after exposure to the polychlorinated biphenyl (PCB) mixture Aroclor 1248

The glutathione-S-transferases (GSTs) from the Northern quahog (Mercenaria mercenaria) were examined after an injection of a polychlorinated biphenyl (PCB) mixture, Aroclor 1248, to a concentration of ~50 ppm. Enzymatic analysis indicated a fourfold increase in the GST activity of quahogs injected with PCBs compared with that of the control. An electrophoretic analysis of the GST from the PCB-exposed quahogs showed a 1.5-fold increase in the concentration over that of the control. Purification of the GST on a glutathione affinity column yielded a glutathione binding protein, in addition to the GSTs. However, the amount of the glutathione binding protein in the PCB-injected quahogs was found to decrease by ~50% in comparison to the glutathione binding protein in the control quahogs.     

Expression in Escherichia coli of Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostii RHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.     

Comparative effects of commercial Aroclors on rat liver enzyme activities

Hepatic DNA, RNA, protein and p-nitroanisole O-demethylase, aniline hydroxylase, nonspecific carboxylesterase, bromosulphophthalein-glutathione (BSP-GSH) conjugating enzyme and p-nitrophenol UDPglucuronyl transferase activities were measured in young Wistar male rats which had received intraperitoneal injections (50 mg/kg) of biphenyl and Aroclors 1016, 1221, 1232, 1242, 1248, 1254 and 1260, dissolved in peanut oil, for 3 consecutive days and assayed 96 h after the last injection. Biphenyl and all the Aroclors caused the same degree of enhancement of BSP-GSH conjugating enzyme. Decreased DNA content, increased RNA and protein content and the other enzymatic activities were related to the percent weight of chlorine and the chlorobiphenyl composition of the Aroclors. More marked effects were observed with the highly chlorinated Aroclor 1248, 1254, and 1260 mixtures which contained predominantly tetra-, penta-, hexa-, and higher-chlorinated biphenyls.     

Purification and properties of ferredoxinBPH, a component of biphenyl 2,3-dioxygenase of Pseudomonas sp strain LB400

The ferredoxin component (ferredoxin_BPH) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filtration column chromatography. The protein was a monomer with a molecular weight of 15000 and contained 2 gram-atoms each of iron and acid-labile sulfur. Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm. The spectrum was partially bleached in the visible region upon reduction by reductase_BPH with NADPH as the source of electrons. Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein. Upon reduction with sodium dithionite, signals with g_x = 1.82, g_y = 1.92 and g_z = 2.02 were detected. These results indicate that the protein contains a Rieske-type (2Fe-2S) iron-sulfur center. Ferredoxin_BPH was required for the oxidation of biphenyl by the terminal oxygenase component of the enzyme and is probably involved in the transfer of reducing equivalents from reductase_BPH to the terminal oxygenase during catalysis. Received 01 November 1996/ Accepted in revised form 27 May 1997     

Studies on metabolic activation of vinyl chloride in Drosophila melanogaster after pretreatment with phenobarbital and polychlorinated biphenyls.

It is known that vinyl chloride is metabolized by the mixed function oxygenase system in the liver to reactive mutagenic and carcinogenic metabolites. This metabolic activation was studied in Drosophila melanogaster by measuring the uptake of 14C from labelled vinyl chloride in different strains and with different pretreatments with phenobarbital and polychlorinated biphenyl (PCB) Clophen A50), well known inducers of cytochrome P-450. In accordance with previously obtained data on vinyl chloride induced sex linked recessive lethals, it was shown that pretreatment with inducers increased the uptake of labelled compound up to ten times. There was, however, a marked difference in response between the five strains used. In particular, the strain Hikone, known to be resistant to insecticides, had a comparatively high initial radioactivity from vinyl chloride without any pretreatment, but it was not or insignificantly inducible with phenobarbital or PCB. Crosses between Hikone and an inducible strain indicated essentially a dominance for the Hikone genotype. Tests on inducible strains showed the same response to phenobarbital by 2 h old larvae and adult male and females. Dimethylsulphoxide (DMSO) used as a solvent decreased both the initial uptake of 14C and particularly the induction by PCB. The use of Tween 80 as an emulsifier did not have such an effect. It is emphasized that the interstrain variation in metabolic activation and inducability has to be taken into consideration in order to optimize the use of Drosophila for mutagenicity testing. This variation also opens up new possibilities of analyzing the mixed function oxygenase system biochemically and genetically.     

Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures

The effects of a commercial polychlorinated biphenyl (PCB) mixture (Aroclor 1248) and two individual PCB congeners were evaluated on rat renal proximal tubule culture cell viability and internucleosomal DNA fragmentation (DNA ladder) characteristic of apoptosis. Treatment with Aroclor 1248 caused the loss of cell viability and promoted apoptosis in a concentration- and time-dependent manner. The two PCB congeners assessed can also induce apoptosis. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (3,3,4,4-tetrachlorobiphenyl) than for the di-ortho-substituted nonplanar congener (2,2,4,4,5,5-hexachlorobiphenyl). This correlated with the loss of cell viability since the planar compound is much more cytotoxic. The results suggest a different molecular mechanism in the induction of apoptosis by planar or nonplanar PCB congeners.     

Potent induction of rat liver microsomal, drug-metabolizing enzymes by 2,3,3',4,4',5-hexabromobiphenyl, a component of fireMaster

The multistep synthesis and purification of 2,3,3',4,4',5-hexabromobiphenyl (HBBp) is described. Capillary gas chromatography revealed that HBBp comprises 0.05% of the industrial polybrominated biphenyl (PBB) mixture, fireMaster BP-6 (lot 7062). When administered to immature male Wistar rats, HBBp caused a dose-dependent increase in (a) the activity of benzo[a]pyrene (B[a]P) hydroxylase (AHH) and 4-chlorobiphenyl (4-CBP) hydroxylase and (b) the concentration of cytochrome P-450. Sodium dodecyl sulfate (SDS)-gel electrophoresis indicated that these increases in cytochrome P-450 and cytochrome P-450-dependent monooxygenase activities were accompanied by a dose-dependent intensification of a protein of relative molecular weight (Mr) 55 000 which comigrated with the major 3-methylcholanthrene(MC)-inducible form of cytochrome P-450 (i.e., cytochrome P-448). Like MC, but in contrast to phenobarbitone (PB), HBBp competitively displaced 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]-TCDD) from the cytosolic protein thought to be the receptor for cytochrome P-448 induction. The results indicate that HBBp is a potent inducer of cytochrome P-448 and as such is the third MC-type inducer identified in fireMaster BP-6.