Comparison of DNA adduct induction in vitro by PAHs and nitro-pahs in freshly isolated rat hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs have been identified widely in occupational and environmental pollution, such as diesel engine emissions and other combustion products. In most cases, hepatic biotransformation is involved in converting these chemicals to their carcinogenic metabolites. It has been demonstrated that isolated hepatocytes possess substantial amounts of the enzymes responsible for metabolizing xenobiotics and are therefore a convenient model for studying chemicals that require activation to exert their carcinogenic effects. In this study, rat hepatocytes were isolated by collagenase digestion and then exposed to benzo[a]pyrene (B) [a]P), benzo[a]anthracene (B[a]A), 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-DNP) at different doses and/or times so that DNA adducts levels, as measured with the ³²P-postlabelling technique, could be compared. Each of the four compounds tested induced significant increases of total DNA adducts with clear dose-related responses. One or more individual adducts were identified as major adducts for each compound. Time-related increases of DNA adducts were also observed from 1 to 4 hr of incubation. Greater amounts of DNA adducts were induced by B[a]P or 1,6-DNP than by B[a]A or 1-NP, with potency being in the order 1,6-DNP > B[a]P > 1-NP B[a]A. These results demonstrate that freshly isolated hepatocytes can be used as an effective in vitro system for the detection of DNA adducts using ³²P-postlabelling, and have shown 1,6-DNP to be the most potent of the tested constituents of diesel emissions.     

Effect of a dioxin-like PCB (CB 126) on the biotransformation and genotoxicity of benzo

Two experiments were performed to study the interaction between benzo[a]pyrene (BaP) and the planar, dioxin-like PCB congener 3,3′,4,4′,5-pentachlorobiphenyl (CB 126) in the flatfish dab (Limanda limanda). The first experiment involved four groups. Group I was treated with 10 μg/kg CB 126, group II was treated with 2 mg/kg BaP, group III was first treated with 10 μg/kg CB 126 and exposed to 2 mg/kg BaP 6 days later, and group IV was a control group. The second experiment was similar, except that the BaP dosage level was increased to 50 mg/kg. Pre-treatment with 10 μg/kg of CB 126 always caused the induction of hepatic cytochrome P450 1A (CYP1A), as measured by significant increases of the model reaction 7-ethoxyresorufin-O-deethylase (EROD) in microsomal preparations. Treatment of dab with BaP caused a significant EROD induction at the 50 mg/kg, but not at the 2 mg/kg level. Concurrent with EROD induction by either CB 126 or 50 mg/kg BaP, was a significant change in the biliary metabolite pattern in favour of 1-hydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene and towards a lower fraction of the procarcinogen BaP-7,8-dihydrodiol (7,8-DIOL). Pre-treatment with CB 126 did not cause an increase of hepatic BaP DNA adducts formed after treatment with either 2 or 50 mg/kg BaP. Glutathione S-transferase (GST) activities remained also unaffected by any of the treatments. The results of this study suggest that the pattern of BaP metabolites in bile depends on the level of CYP1A induction. Moreover, the concurrence of a potent CYP1A inducer and BaP does not necessarily lead to an increase in DNA adduct levels in liver tissue. The observation that the level of 7,8-DIOL is decreased despite a higher (CYP1A mediated) EROD activity explains, at least in part, the lack of induction of DNA adducts.     

Multiple opiate receptors: Differential binding of μ, κ and δ agonists

Much data has accumulated suggesting that μ and δ opiate receptors represent distinct topographical units within the CNS. In addition, in vitro binding studies by the present authors also suggest that a unique κ receptor may be present in the brain. In this regard, [³H]ethylketocyclazocine binding to membrane preparations of rat brain was found to be potently displaced by κ partial μ and agonist/antagonist analgesics. However, μ, δ, σ and ε receptor agonists were much less active at this binding site, suggesting that the κ site possesses a different pharmacology from μ and δ sites. These data therefore support previous in vivo experiments which demonstrated a unique behavioral pharmacology and CNS distribution for κ-mediated analgesia.     

Aldo-keto reductase- and cytochrome P450-dependent formation of benzo[a]pyrene-derived DNA adducts in human bronchoalveolar cells

There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) induce lung cancer through metabolic activation. As part of a program to delineate the routes of PAH activation, we have examined DNA adducts that are formed in human lung cells. A stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry method was used to quantify eight anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE)-derived DNA adducts in four H358 human bronchoalveolar cell lines with different phenotypes. In P450 1A1/P450 1B1-induced H358 cells exposed to (+/-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), (+)-anti-trans-B[a]PDE-N2-2'-deoxyguanosine [(+)-anti-trans-B[a]PDE-N2-dGuo] was the major DNA adduct, and it formed with no lag phase. In AKR1A1-transfected H358 cells, (+)-anti-trans-B[a]PDE-N2-dGuo was also the major adduct with a 3 h lag phase before significant adduct formation was detected. In AKR1A1-transfected H358 cells with induced P450 1A1/P450 1B1, (+)-anti-trans-B[a]PDE-N2-dGuo was formed with no lag phase in amounts similar to those in the H358 cells with up-regulated P450 1A1/P450 1B1. Surprisingly, the greatest amount of (+)-anti-trans-B[a]PDE-N2-dGuo was formed in the control H358 cells. Furthermore, (+)-anti-trans-B[a]PDE-N2-dGuo formation was 2-fold higher in (-)-B[a]P-7,8-dihydrodiol-exposed H358 cells when compared with (+/-)-B[a]P-7,8-dihydrodiol-exposed cells. The P450 1A1/1B1 inhibitor 2,4,3',5'-tetramethoxystilbene did not attenuate DNA adduct formation in the control H358 cells, suggesting that another P450 was responsible. These data raise the intriguing possibility that P450 1A1/P450 1B1 and AKR1A1 may be protective against (+)-B[a]PDE-mediated DNA damage.     

Induction and inhibition of cytochrome P450-dependent monooxygenases of rats by fungicide bitertanol

The effects of fungicide bitertanol on cytochrome P450-dependent monooxygenases were studied using rats treated intraperitoneally with the N-substituted triazole for 4 days. Treatment with 10, 25, and 100 mg/kg bitertanol produced 2-, 4-, and 14-fold increases of 7-ethoxyresorufin O-deethylation activity in liver microsomes, respectively. Immunoblot analysis of microsomal proteins revealed that 25 mg/kg bitertanol increased CYP1A1 protein in the liver, kidney, and lung by 10-, 13-, and 17-fold, respectively. Bitertanol produced smaller increases of CYP2B and CYP3A catalytic activity and protein than that of CYP1A1 in liver. RT-PCR analysis of total RNA indicated that bitertanol-induced CYP1A1, CYP2B, and CYP3A mRNA. Additions of 0.01–100 μM bitertanol to liver microsomes from rats treated with 25 mg/kg bitertanol or 3-methylcholanthrene inhibited microsomal 7-ethoxyresorufin O-deethylation activity (IC₅₀ = 0.8 or 0.9 μM). Bitertanol at 100 mg/kg increased liver UDP-glucuronosyltransferase and glutathione S-transferase activities by 2-fold. Bitertanol at 25 mg/kg produced a minor increase in metabolic activation of benzo[a]pyrene by liver S-9 fraction in the Ames mutagenicity test while the increase was blocked by addition of 100 μM bitertanol. These findings show that bitertanol is an inducer of CYP1A1, CYP2B, and CYP3A in vivo and an inhibitor of CYP1A catalytic activity in vitro.     

In vitro dermal absorption of pesticides: IV. In vivo and in vitro comparison with the organophosphorus insecticide diazinon in rat, guinea pig, pig, human and tissue-cultured skin

In vitro skin absorption tests are currently being developed as an alternative to in vivo animal tests for predicting the degree of occupational exposure to pesticides. In the study reported here, in vitro percutaneous absorption tests were conducted with the ¹⁴C-ring-labelled pesticide, diazinon, dissolved in acetone and applied to the dermatomed skin (0.5 mm) of a number of species at a dose rate of 9.5–16.7 μg/cm². Skin permeation was determined for 48 hr after exposure using an in vitro flow-through system. Skin permeation was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution and the percentage recovery obtained in methanol washes of the skin at 48 hr and in skin digests. Listed in decreasing order, the total percentage in vitro dermal absorptions (mean ± SD) obtained by 48 hr after exposure for the five skin types were: 47 ± 3.4% (rat), 36 ± 0.9% (tissue cultured Testskin), 33 ± 2.8% (hairless guinea pig), 20 ± 3.1% (human) and 15 ± 13.1% (pig). The percentage recoveries in soapy water skin washes at 24 hr, in methanol washes and skin digests at 48 hr and of ¹⁴C-labelled volatiles collected in air traps at 48 hr after exposure are reported. Comparative in vivo studies demonstrated 37 ± 0.8 and 24 ± 5.7% recovery of ¹⁴C in the urine of rats (dose rate, 6 μg/cm²) and hairless guinea pigs (dose rate, 5 μg/cm²), respectively, by 14 days after exposure. Total faecal recovery 14 days after exposure was 18 ± 0.4 and 4 ± 0.9% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after exposure demonstrated a total tissue recovery of 0.6 ± 0.1% [¹⁴C]diazinon in rats and 1 ± 0.2% in hairless guinea pigs. The total recovery in skin removed from the dose site at 14 days after exposure was 0.2 ± 0.02% and 0.1 ± 0.05% in rats and hairless guinea pigs, respectively. Recovery of radioactivity from soapy water skin washes conducted at 24 hr after exposure was 21 ± 3.8% for rats and 2 ± 0.1% for hairless guinea pigs. Recovery in skin patches was 23 ± 5.4% and 73 ± 2.9% in rats and hairless guinea pigs, respectively. The in vitro data for dermal absorption of [¹⁴C]diazinon for rats (47 ± 3.4%) and hairless guinea pigs (33 ± 2.8%) were in good agreement with the data observed for rats (56 ± 1.03%) and hairless guinea pigs (28 ± 6.0%) in vivo. This study supported the use of in vitro skin absorption tests as an alternative to in vivo animal testing.     

Excretion of benzo[a]pyrene and metabolites in urine and feces of rats: influence of route of administration, sex and long-term ethanol treatment

The urinary and fecal excretion of benzo[a]pyrene (B[a]P) and its main metabolites were studied after oral and intraperitoneal administration of B[a]P to male and female ethanol-treated and non-ethanol- treated rats. After oral administration of B[a]P more mutagenic compounds as well as B[a]P metabolites were found in feces than after intraperitoneal administration. The excretion of B[a]P metabolites in urine and feces after oral administration were maximal at days 1 and 2 whereas after intraperitoneal administration excretion was maximal at days 2 and 3. In males, the amounts of excreted phenolic metabolites in urine and feces were generally higher than in females. The amounts of mutagenic products in urine and feces of males were also higher than in females after intraperitoneal and oral administration of B[a]P. In urine of female rats that received B[a]P intraperitoneally, a decreased excretion of phenolic metabolites was found after ethanol treatment. In feces of both male and female rats, a decreased excretion of 3-OH-B[a]P was found after ethanol treatment. In this study, the influence of sex and administration route on the excretion of B[a]P metabolites was more pronounced than the effect of ethanol treatment.     

Unscheduled DNA synthesis in tracheal epithelial cell cultures

An in vitro method has been established for the isolation and culture of tracheal epithelial cells for the evaluation of chemically induced genotoxicity using an unscheduled DNA synthesis assay. Cell cultures were derived from the Wistar albino rat and the golden Syrian hamster. Epithelial cells were isolated with protease type XIV for 16 hr and allowed to attach for 24 hr on collagen-coated coverslips in multi-well plates. Cells were exposed to the experimental carcinogens benzo[a]pyrene (metabolism-dependent) and methylmethanesulphonate (direct acting) for 24 hr. Benzo[a]pyrene and methylmethanesulphonate induced DNA repair in cultures isolated from hamsters, whereas only methylmethanesulphonate induced unscheduled DNA synthesis in rat tracheal epithelial cells, thus indicating the lack of metabolic activation in the rat cultures. These results could indicate the suitability of this culture system for the evaluation of airborne carcinogens.     

Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentration

The cytotoxicity of 10 chemicals from the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) list (nos 21–30) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the MEIC programme organized by the Scandinavian Society of Cell Toxicology. The MTT test was used as the endpoint of cytotoxicity after 24hr of exposure to the chemicals. Theophylline, phenobarbital and paraquat were the least cytotoxic compounds in the cellular systems (IC₅₀ = 450-17,000 μm) except for the 3T3 cells. The seven remaining chemicals (dextropropoxyphene, propranolol, arsenic trioxide, cupric sulfate, mercuric chloride, thioridazine and thallium sulfate) showed a similar relative cytotoxic ranking in the four in vitro systems in the lower range of concentrations (IC₅₀ = 2–350 μm). The data suggest that these 10 chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or species specific. The correlation between in vitro data and human lethal blood concentrations showed that the predictability of the in vitro systems was similar to that of in vivo rodent tests (LD₅₀) only when low cytotoxic concentrations (IC₁₀) were used for correlation.     

Temperature-dependent vitellogenin-mRNA expression in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes at 14 and 18 degrees C.

In order to study the influence of temperature on vitellogenin gene and estrogen receptor gene expression in primary hepatocytes from rainbow trout (Oncorhynchus mykiss), cells were exposed to 17β-estradiol, bisphenol-A and nonylphenol for 48 and 96 hr. Induction of vitellogenin-mRNA expression was detected in a non-radioactive dot blot/RNAse protection assay and by RT-PCR. In the dot blot/RNAse protection assay, the estrogenic potentials of bisphenol-A and nonylphenol were about 10⁴- to 10⁵-fold and 10⁵-fold lower than that of 17β-estradiol, respectively. The relative estrogenic potential did not show any difference between 14 and 18°C. In contrast, at 18°C, RT-PCR analysis revealed increased amounts of vitellogenin- and estrogen receptor-mRNA after 12 and 24 hr of exposure to 17β-estradiol, if compared to 14°C. Owing to increased vitellogenin gene expression at 18°C, the sensitivity of primary hepatocytes to 17β-estradiol and bisphenol-A could be increased.     

Free-energy perturbation methods to study structure and energetics of DNA adducts: results for the major N2-dG adduct of benzo[a]pyrene in two conformations and different sequence contexts

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is activated to (+)-anti-B[a]PDE, which induces a variety of mutations (e.g., G --> T, G --> A, etc.) via its major adduct [+ta]-B[a]P-N2-dG. One hypothesis is that adducts (such as [+ta]-B[a]P-N2-dG) induce different mutations via different conformations, probably when replicated by different lesion-bypass DNA polymerases (DNAPs). We showed that Escherichia coli DNAP V was responsible for G --> T mutations with [+ta]-B[a]P-N2-dG in a 5'-TGT sequence (Yin et al., (2004) DNA Repair 3, 323), so we wish to study conformations of this adduct/sequence context by molecular modeling. The development of a CHARMM-based molecular dynamics (MD) simulations protocol with free-energy calculations in the presence of solvent and counterions is described. A representative base-pairing and base-displaced conformation of [+ta]-B[a]P-N2-dG in the 5'-TGT sequence are used: (1) BPmi5, which has the B[a]P moiety in the minor groove pointing toward the base on the 5'-side of the adduct, and (2) Gma5, which has the B[a]P moiety stacked with the surrounding base pairs and the dG moiety displaced into the major groove. The MD output structures are reasonable when compared to known NMR structures. Changes in DNA sequence context dramatically affect the biological consequences (e.g., mutagenesis) of [+ta]-B[a]P-N2-dG. Consequently, we also developed a MD-based free-energy perturbation (FEP) protocol to study DNA sequence changes. FEP involves the gradual "fading-out" of atoms in a starting structure (A) and "fading-in" of atoms in a final structure (B), which allows a realistic assessment of the energetic and structural changes when two structures A and B are closely related. Two DNA sequence changes are described: (1) 5'-TGT --> 5'-TGG, which involves two steps [T:A --> T:C --> G:C], and (2) 5'-TGT --> 5'-TGC, which involves three steps [T:A --> T:2AP --> C:2AP --> C:G], where 2AP (2-aminopurine) is included, because T:2AP and C:2AP retain more-or-less normal pairing orientations between complementary bases. FEP is also used to evaluate the impact that a 5'-TGT to 5'-UGT sequence change might have on mutagenesis with [+ta]-B[a]P-N2-dG. In summary, we developed (1) a CHARMM-based molecular dynamics (MD) simulations protocol with free-energy calculations in the presence of solvent and counterions to study B[a]P-N2-dG adducts in DNA duplexes, and (2) a MD-based free-energy perturbation (FEP) protocol to study DNA sequence context changes around B[a]P-N2-dG adducts.     

Tissue differences,dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L) exposed to benzo[a]pyrene

Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a). to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b). to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c). to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 microg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 microg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 microg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 microg/l, mean+/-SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 microg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 microg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in M. edulis for monitoring purposes, analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.     

Percutaneous penetration of octyl salicylate from representative sunscreen formulations through human skin in vitro

The human skin penetration of [¹⁴C]octyl salicylate from two representative sunscreen vehicles was determined in vitro. ³H-sucrose was incorporated into all formulations and provided a marker for membrane integrity. When applied as a finite dose in an oil-in-water emulsion vehicle containing 5% (w/w) octyl salicylate, the average total absorption of ¹⁴C over 48 hr was 0.65 ± 0.16% of the applied dose (representing a total amount permeated of 1.58 ± 0.36 μg/cm²). When applied as an infinite dose in the oil-in-water emulsion vehicle the average total absorption of ¹⁴C over 48 hr was 0.47 ± 0.22% of the applied dose (representing a total amount permeated of 27.54 ± 13.91 μg/cm²). When applied as a finite dose in a representative hydroalcoholic formulation containing 5% (w/w) octyl salicylate, the average total absorption of ¹⁴C over 48 hr was 0.59 ± 0.09% of the applied dose (representing a total amount permeated of 1.58 ± 0.25 μg/cm²). When applied as an infinite dose in the hydroalcoholic formulation the average total absorption of ¹⁴C over 48 hr was 0.23 ± 0.05% of the applied dose (representing a total amount permeated of 11.28 ± 2.55 μg/cm²). The penetration of [¹⁴C]salicylic acid [applied at a concentration of 2.7% (w/w), in the oil-in-water emulsion] was also determined. When applied as a finite dose the average total absorption of ¹⁴C over 48 hr was 1.14 ± 0.23% of the applied dose (representing a total amount permeated of 1.65 ± 0.39 μg/cm²). These results suggest that the in vitro human skin permeation of octyl salicylate is relatively low. The amounts of octyl salicylate and salicylic acid permeated when applied in similar vehicles were remarkably similar over 48 hr (1.58 μg/cm² and 1.65 μg/cm², respectively). This suggests the possibility that the ¹⁴C label appearing in the receptor fluid may, in both cases, represent salicylic acid. If this is the case, then it is possible that the amount of octyl salicylate permeating through the skin is much less than that suggested by the data obtained here. This supposition is, however, entirely speculative and has yet to be confirmed experimentally.     

Inducibility and functionality of rat embryonic/foetal cytochrome P-450: A study of differentiating limb-bud and mid-brain cells in vitro

The presence of constitutive levels of cytochrome P-450 isoenzymes in cultures derived from rat embryo limb-bud (LB) and mid-brain (CNS) cells was demonstrated immunocytochemically by staining with specific monoclonal and polyclonal antibodies of cytochrome P-450. The b and e forms of cytochrome P-450 were found to be non-inducible by either in vitro co-incubation for 5 days or by transplacental maternal induction with phenobarbitone (PB), 3-methylcholanthrene (3MC) or β-naphthoflavone (βNF) in either cell type. Consistent with this lack of response was the observation that both in vitro and in vivo inducer treatment did not alter the toxicity of the teratogens diphenylhydantoin (DPH) or cyclophosphamide (CPA). In contrast, 3MC induction was achieved by both in vitro and transplacental regimens as gauged by the increased intensity of peroxidase staining using a monoclonal antibody to cytochrome t-450 c, in both cell types. There was also a concomitant increase in DPH toxicity (>20% gauged by a decrease in IC₅₀ values) in LB cells by both induction regimens but the CNS cells were refractory. βNF induction of cytochrome P-450 was observed following in vitro and in vivo exposures in both cell types. There was no modulation of DPH or CPA toxicity after in vitro exposure to the inducers, but in vivo induction caused a strong staining reaction in both cell types, commensurate with a 30% increase in DPH toxicity in LB cells and activation of the pro-teratogen CPA. The b and e forms of cytochrome P-450 were non-inducible but it is highly likely that the c form was both inducible (by 3MC and βNF) and functional, the latter being assessed by modulation of DPH toxicity and CPA activation. It may be possible to induce cytochrome P-450 in cells derived from embryos. The system used may be suitable for detailed investigations of the types of metabolizing systems involved in the mechanisms underlying toxicity/teratogenicity.     

Lack of significant changes in μ, δ opioid binding sites and neutral endopeptidase EC 3.4.24.11 in the brain and spinal cord of arthritic rats

The possible changes in neutral endopeptidase EC 3.4.24.11 (“enkephalinase”, NEP), μ and δ opioid binding sites, were investigated using in vitro quantitative radioautography in various regions of the central nervous system of the Freund's adjuvant-induced arthritic rat, a model of chronic pain. Enkephalinase was labelled by a specific tritiated inhibitor, [³H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-l-oxopropyl]glycine ([³H]HACBO-Gly), while μ and δ opioid binding sites were selectively labelled with [³H]Tyr- -Ala-Gly-(Me)Phe-Gly-ol ([³H]DAGO) and [³H]Tyr- -Thr-Gly-Phe-Leu-Thr ([³H]DTLET), respectively. As compared to controls, no significant modifications were found in NEP, μ or δ binding sites at both supraspinal and spinal levels of arthritic rats. These results suggest that the enhanced efficiency of exogenous opioids or endogenous enkephalins, reported to occur in this model of chronic inflammatory pain, are not directly related to changes in μ and δ opioid binding sites or steady state levels of NEP.     

In vitro dermal absorption of pesticides: VI. In vivo and in vitro comparison of the organochlorine insecticide DDT in rat, guinea pig, pig, human and tissue-cultured skin

In vitro dermal absorption tests were conducted with the ¹⁴C-ring-labelled pesticide, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 16–27 μg/cm². Skin absorption was determined for 48 hr after exposure using in vitro flow-through cells. Skin absorption was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution added to the percentage recovery for the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline (used with Moody aluminium cells), and Hanks' HEPES buffered saline with 4% serum albumin (used with Bronaugh flow-through cells) were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 42 ± 2.6% [hairless guinea pig; Hanks' receiver (HR)], 34 ± 10.5% (rat; HR), 28 ± 13.2% [Testskin; Ringer's receiver (RR)], 28 ± 2.9% (human; HR), 22 ± 3.3% (Testskin; HR), 18 ± 6.2% (pig; RR) and 14 ± 2.1% (pig; HR). The percentage ¹⁴C activity recovered in soapy water rinses of the skin specimens at 24 hr, and for methanol skin washes and skin digests at 48 hr, and of ¹⁴C-labelled volatiles collected in air traps are reported. Data obtained with pig and Testskin for DDT using the Moody flow-through permeation cell was compared with that obtained using the Bronaugh cell. Significantly greater (P < 0.05) percentage recovery was obtained for the soap washes at 24 hr of the skin following the Bronaugh procedure than was obtained with the Moody method. Comparative in vivo studies demonstrated urinary recovery was 2 ± 0.5 and 15 ± 1.7% for rats (dose rate; 6 μg/cm²) and guinea pigs (dose rate: 9 μg/cm²), respectively. Total faecal recovery was 20 ± 1.9 and 44 ± 2.75% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after dosing demonstrated total tissue recovery of 51 ± 5.6% in rats but of only 3 ± 0.7% in guinea pigs. Including the ¹⁴C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 73 ± 5.9 and 62 ± 4.1% for rats and guinea pigs, respectively. Recovery of ¹⁴C from soapy water skin washes conducted at 24 hr after exposure was 3 ± 1.4 and 14 ± 1.8% for rats and guinea pigs, respectively, and this was significantly less than that obtained by both the Bronaugh and Moody in vitro procedures. Skin patch recovery was 24% for both rats and guinea pigs. In summary, the in vitro data underestimed the degree of dermal absorption observed in vivo for both rats and guinea pigs, and this was thought to be due to an overly vigorous removal of the pesticide skin deposit by the soap washing procedures used in vitro in comparison with the in vivo washing procedure.     

Persistence of benzo[a]pyrene--DNA adducts in hematopoietic tissues and blood of the mummichog, Fundulus heteroclitus

The formation and persistence of benzo[a]pyrene (B[a]P)-DNA adducts were investigated in blood, liver and two hematopoietic tissues (anterior kidney and spleen) of the mummichog (Fundulus heteroclitus). Fish were injected with a single, sublethal dose of B[a]P (12 mg/kg body weight) and sampled from 8 to 96 days post-injection. 32P-Postlabeling analysis and storage phosphor imaging were used to resolve and quantify hydrophobic DNA adducts. One major DNA adduct was present in each of the examined tissues at all sampling times. This adduct had similar chromatographic characteristics to those of the adduct standard, 7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydro-benzo[a]pyrene (B[a]PDE-dG). Minor DNA adduct spots, representing less than 2% of the total DNA adducts, were observed in some liver, anterior kidney and spleen samples for up to 32 days post-injection. The B[a]P-DNA adducts reached maximal levels at 32 days post-injection and persisted for at least 96 days in all examined tissues. B[a]P-DNA adduct levels were significantly higher in the liver and anterior kidney than in the spleen from 16 to 96 days (P<0.001), although liver and anterior kidney DNA adduct levels were not significantly different at any time. This is the first controlled study to demonstrate the formation and persistence of B[a]P-DNA adducts in hematopoietic tissues and blood of fishes exposed to the prototypical polycyclic aromatic hydrocarbon, B[a]P. Although persistent DNA adducts are generally recognized as potential initiators of carcinogenic processes, adducts in these vital tissues may also lead to disruption of physiological functions such defense mechanisms and hematopoiesis.     

Increased peroxisomal enzyme mRNA levels in adult rat hepatocytes cultured in a chemically defined medium and treated with nafenopin

Nafenopin is a known inducer of peroxisome proliferation in the hepatocytes of treated rodents. Primary cultures of adult rat hepatocytes maintained in a chemically-defined medium respond to the drug. RNAs from hepatocyte cultures treated for 1, 8 and 20 hr and their untreated counterparts have been purified and hybridized to radioactive cDNA probes specific for peroxisomal mRNAs (for catalase and the three enzymes of the β-oxidation system). The amount of the specific mRNAs was fairly constant or increased slightly in control cultures, but increased steadily during treatment of the cultures with a non-toxic dose of nafenopin (32 μ ). For the peroxisomal bifunctional enzyme mRNA, representative of the β-oxidation system, this increase was approximately fivefold after 20 hr, whereas for catalase mRNA a twofold increase compared with the control was observed after 20 hr. The time-course of the induction of the peroxisomal bifunctional enzyme mRNA in vitro was found to be similar to that observed after intragastric treatment of rats with nafenopin. This indicates that mechanistic studies on the early events induced in hepatocytes by peroxisome proliferators can be performed with this culture system. Such studies may help to explain the hepatotoxic/hepatocarcinogenic properties of this class of xenobiotics.     

In vitro dermal absorption of pesticides: V. In vivo and in vitro comparison of the herbicide 2,4-dichlorophenoxyacetic acid in rat, guinea pig, pig, human and tissue-cultured skin

In vitro dermal absorption tests were conducted with the ¹⁴C-ring-labelled herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 7–8 μg/cm². Skin absorption was determined for 48 hr after exposure using an in vitro flow-through system. Skin absorption was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution and the percentage recovery in the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline, and Hanks' HEPES buffered saline with 4% serum albumin were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 47 ± 4.3% [tissue cultured Testskin; Hanks' receiver (HR)], 40 ± 4.5% (rat; HR), 19 ± 1.8% (human; HR), 14 ± 2.3% (hairless guinea pig; HR), 14 ± 8.8% (pig; Ringer's receiver). The percentage recovery of the radiolabel in soapy water skin washes at 24 hr, methanol washes and skin digests at 48 hr, and of ¹⁴C-labelled volatiles collected in air traps at 48 hr after exposure are reported. Comparative in vivo studies were conducted for 14 days after exposure and demonstrated 32 ± 3.9 and 28 ± 7.8% recovery of ¹⁴C in the urine of rats (dose rate, 3 μg/cm²) and guinea pigs (dose rate, 4 μg/cm²), respectively. Total faecal recovery was 2 ± 0.3 and 9 ± 3.5% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 day after dosing demonstrated a total tissue recovery of ¹⁴C activity of 1 ± 0.1 and 2 ± 0.5% in rats and guinea pigs, respectively. Including the ¹⁴C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 49 ± 10.4 and 40 ± 9.9% in rats and guinea pigs, respectively. Recovery of ¹⁴C activity from soapy water skin washes conducted at 24 hr after exposure was 28 ± 8.1 and 43 ± 9.0% for rats and guinea pigs, respectively. Recovery in skin patches was 18% (guinea pigs) and 26% (rats). In summary, the in vitro/in vivo concordance for the rat dermal absorption data was good but the in vitro data for hairless guinea pigs underestimated the in vivo absorption, and therefore for 2,4-D, rat skin may provide a better model of percutaneous absorption.     

The effects of morphine, cocaine, amphetamine and hashish on the phagocytosis of the protozoon Tetrahymena pyriformis strain W

Cells of the ciliated protozoon Tetrahymena pyriformis, strain W, grown in a peptone-yeast medium usually contain many phagocytic vacuoles. The phagocytic activity of this protozoon was studied in vivo using heat-killed yeast stained with carmine dye and after exposing the cultures for 2 hr to morphine (20 μg/ml), cocaine (20 μg/ml), amphetamine (0.5 μg/ml) or hashish (0.1 μg/ml). The number of vacuoles formed indicated the phagocytic activity after treatment with the drugs of abuse. Amphetamine caused a slight increase (P < 0.05) in the phagocytic activity of the protozoon, whereas morphine, cocaine and hashish each caused a significant (P < 0.01) decrease in this activity.