Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse

FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.     

Analysis of gene mutations and clastogenicity following short-term treatment with azathioprine in MutaMouse.

The mutagenicity and clastogenicity of the immunosuppressive drug azathioprine (AZA), a multitissue rodent carcinogen and IARC-classified human carcinogen, was investigated using transgenic lacZ mice (MutaMouse). Male animals (n = 5 per group) were dosed with AZA (10, 50, 100 mg/kg p.o. daily for 5 days), vehicle (n = 10), or the positive control, chlorambucil (15 mg/kg i.p., n = 3), and killed 24 hr or 25 days after the last treatment. Micronucleus assays were performed with bone marrow (24-hr samples) or peripheral blood (24-hr and 25-day samples) and DNA was extracted from bone marrow and liver for gene mutation analysis at the transgenic lacZ locus. AZA induced 5.3-111.3-fold increases in %MNPCE (P < 0.01) in bone marrow compared with vehicle control, accompanied by 4.4-5. 6-fold increases in %MNRETs (P < 0.01) in peripheral blood. Chlorambucil caused a 14.5-fold increase in %MNRET and there was evidence of significant stem cell toxicity in both positive control and AZA treatment groups. By day 25, however, there was evidence of substantial recovery of the bone marrow as determined by the frequency of RET, and the %MNRET in all treatment groups was the same as the vehicle control. Analysis of lacZ MF showed 1.4-1.6-fold increases in AZA 24-hr bone marrow samples, increasing to approximately 2.0-fold above concurrent controls by day 25 (medium dose P < 0.05, high dose P < 0.01). For liver, there was a 2-fold increase in MF (P < 0.05) in the 24-hr sample at the highest dose only, and increases of 1.3-1.5-fold by day 25 in the medium (P < 0. 05) and high (P = 0.055) dose groups, respectively. The positive control, chlorambucil, induced 2-3-fold increases (P < 0.01) in mean MF in both bone marrow (25-day sample) and liver (24-hr and 25-day samples). These data confirm the clastogenicity of AZA in the mouse, and show that this compound induces gene mutations in bone marrow and liver, in vivo, at the highest dose and supports the view that AZA is a genotoxic carcinogen.     

Tissue-specific metabolic activation and mutagenicity of 3-nitrobenzanthrone in MutaMouse

3-Nitrobenzanthrone (3-NBA) is a mutagen and suspected human carcinogen detected in diesel exhaust, airborne particulate matter, and urban soil. We investigated the tissue specific mutagenicity of 3-NBA at the lacZ locus of transgenic MutaMouse following acute single dose or 28-day repeated-dose oral administration. In the acute high dose (50 mg/kg) exposure, increased lacZ mutant frequency was observed in bone marrow and colonic epithelium, but not in liver and bladder. In the repeated-dose study, a dose-dependent increase in lacZ mutant frequency was observed in bone marrow and liver (2- and 4-fold increase above control), but not in lung or intestinal epithelium. In addition, a concentration-dependent increase in mutant frequency (8.5-fold above control) was observed for MutaMouse FE1 lung epithelial cells exposed in vitro. 1-Nitropyrene reductase, 3-NBA reductase, and acetyltransferase activities were measured in a variety of MutaMouse specimens in an effort to link metabolic activation and mutagenicity. High 3-NBA nitroreductase activities were observed in lung, liver, colon and bladder, and detectable N-acetyltransferase activities were found in all tissues except bone marrow. The relatively high 3-NBA nitroreductase activity in MutaMouse tissues, as compared with those in Salmonella TA98 and TA100, suggests that 3-NBA is readily reduced and activated in vivo. High 3-NBA nitroreductase levels in liver and colon are consistent with the elevated lacZ mutant frequency values, and previously noted inductions of hepatic DNA adducts. Despite an absence of induced lacZ mutations, the highest 3-NBA reductase activity was detected in lung. Further studies are warranted, especially following inhalation or intratracheal exposures.     

Novel transgenic rat for in vivo genotoxicity assays using 6-thioguanine and Spi- selection

Transgenic rodents are valuable models for investigating the genotoxicity of chemicals in vivo. Here, we report the establishment of a novel transgenic rat for genotoxicity analysis. In this model, about 10 copies of lambdaEG10 DNA carrying the gpt gene of E. coli and the red/gam genes of lambda phage are integrated per haploid genome of Sprague-Dawley rats at position 4q24-q31. After recovery of lambdaEG10 phage, point mutations in the gpt gene and deletions in the red/gam genes are identified by 6-thioguanine and Spi(-) selection, respectively. To examine the suitability of these rats for performing in vivo mutagenicity assays, rats were treated with single intraperitoneal injections of ethylnitrosourea (ENU; 100 mg/kg) or benzo[a]pyrene (B[a]P; 62.5 and 125 mg/kg), and the mutant frequencies (MFs) in the liver were determined 7 days after the treatment. ENU enhanced the gpt MF about 7-fold over the control while it did not significantly increase the Spi(-) MF. B[a]P increased both the gpt and Spi(-) MFs several-fold in a dose-dependent manner. To examine the kinetics of MF, ENU was administered (50 mg/kg/day for 5 successive days) and gpt MFs in the liver were determined 7, 21, 35, and 70 days after the last injection. The MF increased to 8-fold and 13-fold over the control at 7 and 35 days, respectively, after the last injection and then slightly declined at 70 days. These kinetics are similar to those reported for ENU-treated lacZ transgenic mice. This novel transgenic rat could be useful for investigating species differences between rats and mice in their response to genotoxic agents.     

Partial hepatectomy strongly increased the mutagenicity of N-ethyl-N-nitrosourea in MutaMouse liver.

The relationship between cell proliferation and mutagenesis can be investigated in vivo due to the advent of transgenic animal mutation assays. In these assays, slowly proliferating tissues, such as mammary gland and liver, that are exposed to mutagens generally have longer manifestation times for mutations and lower mutant frequencies. This may be because the cells have enough time prior to cell division to repair DNA damage. We carried out this study using the MutaMouse positive selection system to investigate the effect of a high rate of cell proliferation induced by partial hepatectomy (PH) on mutation induction. We used a 2 x 2 experimental design for PH (or no PH) and 50 mg/kg N-ethyl-N-nitrosourea (ENU) (or phosphate buffer), with the chemical injected 16-19 hr after PH. In the non-ENU groups, the mean MF was slightly but not significantly higher in the PH group than in the non-PH group. In the ENU non-PH group, the MF was also slightly but not significantly increased. In the ENU PH group, in contrast, the mean MF was 7 times the mean MF of the group that received either treatment alone. These results strongly support the hypothesis that ENU induced pre-mutational DNA lesions in liver are completely repaired prior to cell division, and PH increases the mutagen-induced MF by reducing the amount of time available for such repair.     

Effects of nickel chloride and diethyldithiocarbamate on metallothionein in rat liver and kidney

Effects of NiCl2 and sodium diethyldithiocarbamate (DDC) upon metallothionein (MT) concentrations were studied in liver and kidney of male Fischer rats. After injection of NiCl2 (0.75 mmol per kg, sc), hepatic MT concentration increased 2.6-fold at 6.5 hr and 8.2-fold at 17 hr; renal MT concentration increased 1.4-fold at 6.5 hr and 2.3-fold at 17 hr. Dose-related increases of MT concentrations were observed in liver and kidney of rats killed 17 hr after injection of NiCl2 (0.25 to 0.75 mmol per kg, sc). Repeated administration of NiCl2 (0.1 mmol per kg, ip) on four successive days, with sacrifice three days after the last treatment, increased MT concentrations 1.4-fold in liver and kidney, whereas CdCl2-treatment at the same dosage schedule increased MT concentration 16-fold in liver and 3.3-fold in kidney. NiCl2-Induction of MT in liver and kidney was not prevented by actinomycin D (1 mg per kg, ip), but was inhibited by cycloheximide (2 mg per kg, ip). Sodium diethyldithiocarbamate given alone (1.33 mmol per kg, im) 17 hr before death, increased MT concentration 7.6-fold in liver but did not affect MT concentration in kidney; administration of DDC prior to injection of NiCl2 did not inhibit NiCl2-induction of MT.     

Relative activities of methyl methanesulphonate (MMS) as a genotoxin,clastogen and gene mutagen to the liver and bone marrow of Muta™Mouse mice

The mutagenicity of the rodent carcinogen methyl methanesulphonate (MMS) to the liver and bone marrow of Muta™Mouse lacZ⁻ transgenic mice was evaluated. A single intraperitoneal (i.p.) dose of 100 mg/kg MMS gave a strong positive response in the liver UDS and bone marrow micronucleus assays conducted 2 hr and 30 hr, respectively, after dosing. A single i.p. administration of 100 mg/kg of MMS, or five daily administrations of 20 mg/kg MMS, failed to increase significantly the lacZ⁻ → lacZ⁺ mutation frequency (MF) in either the liver or the bone marrow, albeit some evidence of weak mutagenicity was observed for the liver. The gene mutation analyses were undertaken 14 days after the final chemical exposure. Administration of the liver mitogens dimethylnitrosamine (DMN), or 4-acetylaminofluorene (4AAF), subsequent to multiple (five) exposures of 20 mg/kg MMS, failed to enhance the mutagenicity of MMS to the liver, thereby eliminating the possibility that MMS produced promutagenic lesions in the liver that were not transformed to mutations because of the absence of MMS-induced cell division. In the latter experiments, DMN gave a strong mutagenic response and 4AAF a weak mutagenic response. Possible reasons for this selective mutagenicity of MMS (DNA damage and micronuclei induction in the absence of gene mutations) are discussed, but no clear outcome emerges. It is concluded that transgenic mutation assays should not be employed for defining genetic toxicity in vivo, but rather should be reserved for mechanistic studies on previously established rodent genotoxins and/or carcinogens. Environ. Mol. Mutagen. 32:163–172, 1998 © 1998 Wiley-Liss, Inc.     

Aspects of the purine nucleoside phosphorylase (PNP) deficient state produced in normal rats following oral administration of 8-amino-9-(2-thienylmethyl)guanine (PD 119,229), a novel inhibitor of PNP

8-Amino-9-(2-thienylmethyl)guanine (PD 119,229; 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purine-6-one monohydrochloride) is a potent inhibitor of human purine nucleoside phosphorylase (PNP). The effects of orally administered PD 119,229 on the plasma concentration of the PNP substrates, inosine and guanosine, were determined using normal rats. In time course studies following administration of a single 3 mg/kg dose of PD 119,229, both inosine and guanosine were statistically significantly elevated as soon as one hr postdose. Plasma inosine elevation was maximal ten hr after dosing, reaching a mean of 2.13 M (14.2-fold vehicle). Guanosine was maximally elevated at three hr following a single 3 mg/kg oral dose, reaching a mean of 0.77 M (4.5-fold vehicle). In dose-response studies in which blood specimens were obtained one hr following oral administration of PD 119,229 at doses of 1.5 to 50 mg/kg, maximal mean inosine elevation (1.71 M or 57-fold vehicle) occurred at 15 mg/kg, with a plateauing or decline in inosine concentration noted at higher doses. The maximal mean plasma guanosine concentration was achieved at 50 mg/kg (mean of 0.2 M, or 6.7-fold vehicle). Substantially greater nucleoside elevation was not observed following multiple oral 15 mg/kg doses, nor when nucleoside levels were assessed at two to four hr following a large oral dose. However, inosine levels reaching 15 M, and guanosine concentrations approaching 2 M, were occasionally noted in individual rats. It is concluded that oral administration of PD 119,229 can simulate, in normal rats, one of the more readily detectable biochemical abnormalities of the PNP deficient state in humans.     

In Utero Exposure to Benzo[a]pyrene Induces Ovarian Mutations at Doses That Deplete Ovarian Follicles in Mice

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7–16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410–420, 2019. © 2018 Her Majesty the Queen in Right of Canada     

Sodium phenobarbital-enhanced mutation frequency in the liver DNM of lacz transgenic mice treated with diethylnitrosamine

To investigate how a carcinogenic promoter acts on cells mutatedby an initiator, we used as a model, lacZ transgenic mouse anda positive selection system. Preliminary data for the mutationalevents in liver DNA of the mice was generated using diethylnitrosamine(DEN) and sodium phenobarbital (S-PB) as initiator and promoter,respectively. In our first experiment, male Muta^TMice receiveda single i.p. injection of saline or 100 mg/kg DEN and werefed a normal diet for 7 days and 500 p.p.m. S-PB in the dietfor 21 days. Liver DNA was harvested after a 1 night fast ondays 7 and 28 post-DEN treatment. In our second experiment,male mice received a single i.p. injection of phosphate bufferedsaline or 50 mg/kg DEN and were fed a normal diet for 7 days,a diet with S-PB for 14 days and then a normal diet for 7 days.Liver DNA was harvested after a 1 night fast on days 7, 21 and28 post-DEN treatment. The S-PB diet enhancedabsolute and relativeliver weights in all groups. The single intraperitoneal doseof 50 or 100 mg/kg DEN induced high mutation frequencies (MF)in liver, lacZ genes on days 7, 21 and 28. There were no remarkabledifferences of the MF among any sampling days for animals receivingDEN and a normal diet. S-PB feeding at 500 p.p.m. for 21 daysfailed to affect the MF in groups given saline or 100 mg/kgDEN. On the other hand, when 50 mg/kg DEN was given, S-PB feedingat 500 p.p.m. for 14 days elevated the MF in liver DNA on days21 and 28 to –1.8 and 4.0 times the MF, respectively,ofthe mice fed the normal diet. Consequently, S-PB might preferentiallypromote certain initiated cells participating in a balance betweencell death and proliferation. ¹To whom correspondence should be addressed     

Validation studies with Muta Mouse: a transgenic mouse model for detecting mutations in vivo.

MutaMouse is a transgenic mouse engineered to detect mutations in vivo in any tissue of choice by using simple laboratory methods. The target is a bacterial lacZ gene incorporated via lambda phage into the genome of each mouse cell such that a concatamer of approximately 40 copies exists at a single site on both chromosomes of a homologous pair. In order to assess the potential usefulness of MutaMouse in detecting in vivo mutagenesis, several known mutagens/carcinogens were applied to male animals of 8-10 weeks in age. Intraperitoneal injections (single or 5 daily doses) of N-ethyl-N-nitrosourea (ENU), chlorambucil, procarbazine, cyclophosphamide, and acrylamide were investigated for mutagenic effects in bone marrow, liver, and testes. In addition, skin painting studies (single application) were performed with dimethylbenzanthracene (DMBA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and acetic acid. Increases in mutant frequency were clearly induced by all eight chemicals, the magnitudes of which were dependent on the chemical, dose, method of dosing, tissue analyzed, and the time lapse between treatment and isolation of DNA. Data on variability in mutant frequency was presented relative to the analyzed population of lacZ genes and number of animals per treatment group. Application of the MutaMouse model to the detection of heritable mutations was discussed.     

Detection of gene mutation in skin, stomach and liver of MutaTMMouse following oral or topical treatment with N-methyl-N'-nitro-N-nitrosoguanidine or 1-chloromethylpyrene: some preliminary observations

Transgenic mouse assays, such as Muta^TMMouse, provide a methodto predict the potential target organ carcinogenicity of chemicalcompounds. As part of a validation study, the effects of thedirect-acting mutagens, N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and 1-chloromethyl-pyrene (CMP), were investigated forgene mutation in the tissues of Muta^TMMice after a single oralor topical exposure. MNNG (50 or 100 mg/kg) or CMP (25 or 50mg/kg) were administered as a single oral dose and the micekilled after 3, 7 or 10 days. Mutation frequencies were determinedin stomach DNA from both MNNG and CMP-treated animals and inliver DNA from the MNNG-treated animals only. The results, althoughobtained from a limited number of animals, consistently showedthat MNNG increased the mutation frequency in stomach DNA, butnot apparently in liver DNA, at each exposure time; no clearincrease in mutation frequency was seen in the stomach DNA ofCMP treated animals. Also, MNNG (250 or 500 µg) or CMP(5 or 10 µg) in acetone were applied as a single doseto the shorn skin of mice 7, 14 or 21 days prior to death. Apositive control group was similarly given dimethyl-benz[a]anthracene(DMBA, 40 µg) and sacrificed after 14 days. Mutation frequencieswere determined in the skin DNA extracted from all animals andin the stomach DNA from MNNG-painted animals only. The results,again obtained from a limited number of animals, clearly showedthat all test compounds consistently increased the mutationfrequency of skin DNA and that these increases were far greaterin the DMBA- and MNNG-treated mice than the CMP-treated mice.No apparent increases were seen in the stomach DNA from theMNNG-painted mice. ¹To whom correspondence should be addressed     

Serum and urine concentrations of oral bromovinyldeoxyuridine in humans as monitored by a bioassay system based on varicella-zoster virus focus inhibition

A simple and sensitive bioassay method for measuring (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) concentrations in human serum and urine has been established. This method is based on the inhibitory effect of BVDU on varicella-zoster virus (VZV) focus formation in vitro. The minimal concentration of BVDU that could be detected in serum by this method was 0.2 microgram/ml. Following a single oral administration of 250 mg BVDU, serum BVDU concentrations of 1.2-2.2 micrograms/ml were attained 1 hr later; at 5 and 7 hr, serum BVDU levels were below 0.2 microgram/ml. Upon repeated administration of 125 mg BVDU at 8 hr intervals, the serum BVDU concentrations reached 0.7-1.1 microgram/ml at 2 hr after the fourth administration. These concentrations are approximately 300-450-fold higher than the 50% inhibitory dose of BVDU for VZV in vitro. Urinary BVDU concentrations were on average 10 to 20 times higher than the serum BVDU concentrations.     

Mutagenic activity of cisplatin in the lacZ plasmid-based transgenic mouse model

Cis-diamminedichloroplatinum (II) (cisplatin) is a well characterized antitumor drug used for the treatment of a variety of human cancers. The cytotoxicity of cisplatin is mainly mediated through the formation of DNA adducts, which are also believed to be responsible for the secondary malignancies produced by the drug. The aim of this study was to determine the in vivo mutagenic activity of cisplatin in the lacZ plasmid-based transgenic mouse model. The mutant frequency (MF) and the spectrum of mutations induced by cisplatin in the mouse liver were analyzed and compared to controls. The mean MF in the lacZ gene was increased 2-fold in mice treated with a single 6 mg/kg body weight dose of cisplatin and sacrificed after 17 and 28 days (P = 0.001 and P < 0.0001). Restriction analysis and sequencing of mutant DNA showed that cisplatin was able to induce both large deletions and point mutations. A specific profile of base substitution and frameshift mutations was identified in treated mice, consisting primarily of G:C-->A:T transitions at GpG and ApG sites, the preferential DNA binding sites of cisplatin, and single basepair deletions/insertions. The present results provide the first evidence that cisplatin has mutagenic activity in vivo and induces a characteristic pattern of mutations in the mouse liver. This mutagenicity may be responsible for its tumorigenic activity.     

The cII locus in the MutaMouse system.

Here, we report the first application and characterization of the cII locus as a mutational target for use with the Muta(trade mark)Mouse system for quantifying somatic mutations in vivo. This locus can be analyzed for mutations using positive selection and is identical in sequence to the cII in the Big Blue((R)) Mouse. The cII displays similar spontaneous (5.5 x 10(-5)) and induced mutation frequencies when compared to the lacZ gene in the small intestine of MutaMice treated with ENU (N-ethyl-N-nitrosourea). After acute treatment with 250 mg/kg ENU (ip) the mutant frequencies were 127 x 10(-5) at the cII and 147 x 10(-5) at the lacZ loci, reaching a maximal mutant frequency 10 days posttreatment and remaining constant thereafter. These data prove that this transgene is genetically neutral, conferring neither selective advantage nor disadvantage on the host cells. The cII dose response curve was linear (R(2) = 0.93) comparable to the lacZ after treatments with 0, 50, 150, or 250 mg/kg ENU. Use of the cII locus (0.3 kb) addresses the single most significant drawback associated with the MutaMouse system, namely the inability to obtain sequence spectra efficiently, due to the large size of the lacZ gene (3.0 kb). Moreover, a less obvious application, but nevertheless of considerable importance, is the easy identification of jackpot mutations, without sequencing. The cII, identical in both sequence and origin on the transgenic constructs used in producing the Big Blue and MutaMouse systems, provides the first transgenic locus common to the two widely used in vivo mutagenesis assays.     

Pharmacokinetics of doxycycline in broiler chickens

Doxycycline was given to two groups of eight chickens at a dose of 20 mg/kg of body weight, intravenously (i.v.) or orally. Plasma concentration was monitored serially for 12 h after each administration. Another group of 30 chickens was given 20 mg/kg orally every 24 h for 4 days, and plasma and tissue concentrations determined serially after the last administration. Concentrations of doxycycline were measured using high-performance liquid chromatography. Pharmacokinetic variables were calculated, using a two-compartment open model. The elimination half-life and the mean residence time for plasma were 6.03 +/- 0.45 and 7.48 +/- 0.38 h, respectively, after oral administration and 4.75 +/-0.21 and 2.87 +/-0.11 h, respectively, after i.v. administration. After single oral administration, doxycycline was absorbed rapidly, with T(max) of 0.35 +/- 0.02 h. Maximum plasma concentration was 54.58 +/- 2.44 mu/ml. Oral bioavailability of doxycycline was found to be 41.33 +/- 2.02%. Doxycycline was widely distributed in tissues and considerable concentrations were found following oral administration of 20 mg/kg on four successive days. The results indicate that doxycycline concentrations were cleared slowly and were at or below the accepted drug tolerance levels in the marker tissues within 5 days after dosing.     

Inhibition by Norakin (triperiden) of Sindbis virus infection in mice

Intranasal (i. n.) infection with 10 LD50 of Sindbis virus caused acute encephalomyelitis and death in ABD2F1 mice 3-7 days post infection (p.i.). Histologic lesions were found in the CNS, pancreas. liver, parotid glands, exorbital lacrimal glands, lymphoid organs and kidneys. Repeated oral administration of the anticholinergic anti-Parkinson drug Norakin protected infected animals from death in a dose-dependent manner when treatment was started prior to but not after virus inoculation. The maximum protective effect was achieved when the drug was administered twice daily at doses of 2.5 or 5.0 mg/kg body mass for at least 56 hr; single injections of the full daily dose were ineffective. Daily doses of greater than or equal to 25 mg/kg body mass had a reduced protective effect or failed to prevent mortality. Administration of Norakin up to doses of 300 mg/kg body mass per day to noninfected ABD2F1 mice were tolerated without obvious clinical or histological signs of illness over a period of 104 hr. Replication of sindbis virus in BHK 21/C13 cells was not inhibited by Norakin concentrations up to 10 micrograms/ml. In Mengo virus-infected mice Norakin did not exert any protective effect within the range of 1.25-50.0 mg/kg body mass when treatment started 1 hr before infection and has been continued twice daily over a period of 104 hr.     

Interferon-inducing activity of dipyridamole in mice

The kinetics of interferon (IFN) production was studied in mice after intraperitoneal (i.p.) and oral administrations of dipyridamole. The substance showed a high IFN-inducing activity when given orally at single doses ranging from 12.5 to 100 mg/kg (1/21.5-1/172 of the single LD50 value): peak titres of 2048-4096 IU/ml in the blood serum were reached at 48 hr; elevated IFN levels persisted until day 5 after administration of the inducer. Significantly lower IFN titres were found after i.p. injection.     

D-galactosamine hepatotoxocity. V. Role of free fatty acids in the pathogenesis of fatty liver

The role of free fatty acids (FFA) in the pathogenesis of fatty liver was investigated in female rats who received a single ip injection of d-galactosamine-HCl, (GalN), 750 mg/kg body weight. Groups of rats were either fasted for 14 hr prior to GalN injection and then fasted for the duration of the experiment or were fed ad libitum prior to and after GalN administration. Plasma FFA were determined in groups of fasting or fed rats sacrificed at intervals between 0 and 24 hr after GalN administration. The results revealed a progressive increase in plasma FFA in GalN-injected fasted rats (0 hr, 0.40 μmol/ml; 24 hr, 1.09 μmol/ml) whereas in fed animals only a modest increase in plasma FFA concentrations occurred. Hepatic triglyceride content was determined in groups of fasted and fed rats at 6 and 24 hr after GalN administration. Hepatic triglycerides, in fasted rats, were increased 7-fold at 6 hr (22.4 ± 8.5 mg/g) and were markedly increased at 24 hr (114.8 ± 18.4 mg/g). In contrast, feeding protected the rats from the development of fatty liver since hepatic triglycerides were only twice controls at 6 hr and 4-fold increased 24 hr after GalN. Ultrastructural studies were performed at 15, 24, and 48 hr after GalN in fed and fasted rats. Electron microscopy disclosed hepatocellular necrosis and profound fat accumulation in the fasted animals; however, feeding afforded marked protection against the development of fatty liver and hepatic injury. The results of these studies indicate that the GalN-induced fatty liver is associated with a sustained elevation of plasma FFA. The increase in plasma FFA can be prevented by feeding the animals prior to and after GalN administration suggesting that a stimulus for FFA elevation may be the alterations in carbohydrate metabolism known to be induced by GalN in rat liver.     

The pharmacokinetics of dihydroxypropyltheophylline: a basis for rational therapy.

Dihydroxypropyltheophylline (dyphylline) was administered in a single dose on 7 different days to 7 normal subjects; 5 mg/kg was given by intramuscular injection and oral tablet; 10 mg/kg was given by these routes and also in 3 different liquid formulations. Dyphylline is rapidly absorbed, and bioavailability is independent of route of administration or formulation. The pharmacokinetics of dyphylline do not appear to be dose-dependent in the dosage range studied. The mean half-life of dyphylline is 2.11 +/- 0.36 hr; because of its short half-life and unproved efficacy, current dosage recommendations for dyphylline must be revised greatly. Further efficacy trials utilizing the pharmacokinetic data from this study are essential.