Urinary Bladder In Dogs Research Articles

Evaluation of a method using Proteus mirabilis and Pseudomonas aeruginosa to experimentally induce dual infection of the urinary bladder in dogs.

To evaluate a method using Proteus mirabilis and Pseudomonas aeruginosa to experimentally induce dual infection of the urinary bladder in dogs. 6 healthy mixed-breed dogs. Dogs were anesthetized, and cystitis was induced by infusing a solution of salicylic acid in ethanol into the bladder, followed by an inoculum containing field isolates of P. mirabilis and P. aeruginosa. Dogs were examined daily for 21 days after induction of cystitis. On day 21, dogs were euthanatized, and urinary bladder, renal pelvis, and prostate specimens were submitted for bacterial culture. After induction of cystitis, all dogs had evidence of thickening of the bladder wall, dysuria, tenesmus, and hematuria. Urinalysis revealed proteinuria, hematuria, and pyuria. All urine samples obtained on day 21 yielded growth of P. mirabilis, but P. aeruginosa was not cultured from any of these samples. Proteus mirabilis was isolated from bladder, renal pelvis, or prostate specimens from 4 dogs; P. aeruginosa was not isolated from any of the tissue specimens. Results suggest that the method used in the present study fails to induce dual infection of the urinary bladder with P. mirabilis and P. aeruginosa. The inability to establish a persistent dual infection with this method may have been a result of insufficient pathogenicity of the Pseudomonas isolate or an inadequacy of the experimental design.

ACTIVITY OF NONPEPTIDE TACHYKININ ANTAGONISTS ON NEUROKININ A INDUCED CONTRACTIONS IN DOG URINARY BLADDER

There is abundant evidence indicating that tachykinin (TK) containing sensory nerves (C-fibers) play an important role in neural regulation of reflex micturition in mammals. Desensitization of urinary bladder C-fibers with resiniferatoxin ameliorates bladder hyperreflexia and incontinence and supports a similar role for C-fibers in man. TK-induced contraction of isolated human urinary bladder smooth muscle appears to be exclusively neurokinin NK2-receptor mediated. Dog urinary bladder contractility to a series of TK-receptor agonists also suggests a predominance of NK2-receptor activation by tachykinins and indicates the dog may provide a useful model for the development of pharmacotherapy for bladder hyperreflexia. We evaluated the activity of the nonpeptide NK-receptor selective antagonists SR 48968 (NK2), SB 223412 (NK3) and CP 99994 (NK1) against neurokinin A (NKA)-induced contractions in isolated dog urinary bladder strips to determine the NK-receptor type which mediates contractile responses to NKA. The dual NK1 and NK2 antagonist MDL 103392 was also tested in this preparation. NKA-induced contractions (pD2 = 8.0) were dose dependently blocked by SR 48968 (pA2 = 8.2 +/- 0.2) while CP 99994 (1.0 microM) and SB 223412 (1.0 microM) were inactive. MDL 103392 was a weak functional antagonist (pKb = 6.1 +/- 0.1) of NKA. Relative activities of SR 48968, CP 99994 and SB 223412 confirm that the NK2-receptor is the mediator of NKA-induced contractions of dog urinary bladder smooth muscle.

Expression of cyclooxygenase-2 in transitional cell carcinoma of the urinary bladder in dogs.

To evaluate expression of cyclooxygenase (COX)-1 and COX-2 in the urinary bladder epithelium of clinically normal dogs and in transitional cell carcinoma cells of dogs. 21 dogs with transitional cell carcinoma of the urinary bladder and 8 dogs with clinically normal urinary bladders. COX-1 and COX-2 were evaluated by use of isoform-specific antibodies with standard immunohistochemical methods. COX-1, but not COX-2, was constitutively expressed in normal urinary bladder epithelium; however, COX-2 was expressed in neoplastic epithelium in primary tumors and in metastatic lesions of all 21 dogs and in new proliferating blood vessels in 3 dogs. Also, COX-1 was expressed in the neoplastic cells. Lack of expression of COX-2 in normal bladder epithelium and its substantial expression in transitional cell carcinoma cells suggest that this isoform may be involved in tumor cell growth. Inhibition of COX-2 is a likely mechanism of the antineoplastic effects of non steroidal antiinflammatory drugs.

Magnetic stimulation: a novel method for inducing evacuation of the neuropathic rectum and urinary bladder in a canine model

Objectives. To study the effect of sacral magnetic stimulation (MS) on the neuropathic rectum and urinary bladder in dogs. It has been demonstrated in a canine model and in humans that sacral MS of both the empty and full rectum effected a significant increase in rectal and vesical pressures. Evacuation of the full rectum, as well as balloon expulsion, were achieved. Methods. In 15 dogs, bilateral pelvic ganglionectomy was carried out, and rectal and vesical denervation was confirmed by bethanechol and atropine testing. Sacral MS of the empty and full rectum and urinary bladder were performed by means of a magnetic stimulator and coil. The electromyographic (EMG) response of the rectus abdominus muscles to sacral MS was recorded. Results. After pelvic ganglionectomy, the rectal, vesical, anal, and urethral pressures revealed a significant decrease (all P <0.05). On sacral MS of the empty rectum and urinary bladder, rectal and vesical pressures increased (both P <0.001) and anal and urethral pressures did not change (both P >0.05). Atropine injection abolished the rectal and vesical response to sacral MS; bethanechol did not. During sacral MS, no EMG activity of the rectus abdominus muscles was recorded. Intermittent sacral MS of the full rectum and urinary bladder raised rectal and vesical pressures (both P <0.001) and effected evacuation. Conclusions. Sacral MS causes evacuation of the neuropathic rectum and urinary bladder in a canine model. The technique is simple, noninvasive, nonradiologic, and cost-effective and has no adverse effects. It is suggested that it be used for evacuation of the neuropathic rectum and urinary bladder in patients with spinal cord injury.

Effects of propiverine hydrochloride (propiverine) on isolated rat and dog urinary bladder

Propiverine is a drug for the treatment of incontinence and pollakiuria. The effects of propiverine on isolated rat and dog urinary bladder were investigated. At doses of 10(-6)-3 x 10(-5) M, propiverine caused both a rightward shift and inhibition of the maximum response in the acetylcholine (ACh) dose-response curve. The pA2 values for rat and dog urinary bladder were 5.97 and 6.62, respectively. At doses of 10(-5)-10(-5) M, propiverine also dose-dependently inhibited KCl (100 mM)-induced contractions. The IC50 values for rat and dog urinary bladder were 3.9 x 10(-6) M and 3.8 x 10(-6) M, respectively. The pA2 value and the IC50 value of terodiline for rat urinary bladder were 6.08 and 6.6 x 10(-6) M, respectively. In contrast, the pA2 value and the IC50 value of oxybutynin for rat urinary bladder were 7.69 and 4.5 x 10(-6) M, respectively, suggesting that oxybutynin exerts an anti-muscarinic effect at doses at which no discernible anti-KCl effect was observed, whereas propiverine and terodiline exerted both effects at the same doses. The inhibitory effect of drugs on the contraction induced by electrical field stimulation was tested. At a dose of 10(-7) g/ml, tetrodotoxin inhibited the contraction of rat and dog urinary bladder by 76.6% and 92.6%, respectively. Propiverine and verapamil dose-dependently inhibited the contractile response induced by electrical field stimulation at doses of 10(-5) M or more and 3 x 10(-6) M or more, respectively. At these concentrations, a marked anti-KCl effect of the drugs on smooth muscle was observed. On the other hand, atropine caused no inhibition of the contractile response in rat urinary bladder at a dose of 3 x 10(-5) M, and it inhibited the contraction in dog urinary bladder by 14.9% at a dose of 10(-5) M. These findings suggest that although propiverine exhibits both anti-muscarinic and anti-KCl effects in isolated rat and dog urinary bladder, the inhibitory effects of propiverine on the atropine-resistant part of contraction may be mainly due to its anti-KCl effect.

Cisplatin treatment of transitional cell carcinoma of the urinary bladder in dogs: 18 cases (1983–1993)

Objective To determine whether cisplatin administered at a dosage of 60 mg/m2 of body surface area, IV, every 21 days, would induce remission of transitional cell carcinoma of the urinary bladder in dogs. Design Retrospective analysis of medical records. Animals 18 dogs with histologically confirmed transitional cell carcinoma of the urinary bladder. Procedure Clinical staging was performed by means of physical examination, contrast cystography or ultrasonography, and thoracic radiography prior to and 42 days after the initiation of cisplatin treatment. Dogs with clinical signs of tumor progression were reevaluated earlier than 42 days in some instances. Complete remission (CR) was defined as complete resolution of measurable tumor. Partial remission (PR) was defined as a ≥ 50% reduction in tumor volume without development of new tumors. Stable disease was defined as &lt; 50% change in tumor volume at 42 days without development of new lesions. Progressive disease (PD) was defined as ≥ 50% increase in tumor volume or development of new tumors at any time. Dogs were reevaluated at 42-day intervals until they had a CR, developed PD, or developed unacceptable adverse effects. Results Three dogs had a PR, 4 had stable disease, and 9 had PD. Tumor response could not be assessed in 2 dogs: 1 dog developed grand mal seizures 3 hours after the first dose of cisplatin was given and was euthanatized; the other dog continued to have clinical signs of urinary tract obstruction and was euthanatized 8 days after the first dose of cisplatin. Four dogs developed renal azotemia that was suspected to be secondary to cisplatin nephrotoxicity. Clinical Implications The cisplatin dosage was higher than that reported in studies of dogs with transitional cell carcinoma of the bladder. Even with this higher dosage, none of the dogs had a CR, and only 3 of 18 had a PR. A more effective, less toxic treatment for transitional cell carcinoma in dogs is needed. (J Am Vet Med Assoc 1996;209:1588–1591)

Radioligand binding, autoradiographic and functional studies demonstrate tachykinin NK-2 receptors in dog urinary bladder.

Tachykinin receptors in the dog bladder were characterized using radioligand binding, functional and autoradiographic techniques. In detrusor muscle homogenates, specific binding of [125l]iodohistidyl neurokinin A (INKA) and [125l]Bolton Hunter eledoisin was reversible, saturable and, to a single class of sites of Kd, 3,6 and 27 nM, respectively. No specific binding of [125l]Bolton Hunter[Sar9, Met (O2)11] substance P occurred. INKA binding was reduced by the peptidase inhibitor bacitracin. The rank potency order of agonists competing for binding of both radioligands indicated interaction at NK-2 sites. NK-2-selective antagonists also competed for INKA binding, with SR 48968, GR 94800, MDL 29913 and the selective agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) showing biphasic binding profiles. Autoradiographic studies revealed specific binding of INKA and [125l]Bolton Hunter eledoisin over detrusor muscle and small arteries. [125l]Bolton Hunter [Sar9, Met (O2)11] SP labeled the intima of arteries and arterioles, but not the detrusor muscle. Tachykinins contracted detrusor muscle strips, with potency order at the carbachol EC15 NKA = kassinin > [Lys5, MeLeu9, Nle10]-NKA(4-10) = neuropeptide gamma = neuropeptide K = NKB > > MDL 28564, with [Sar9, Met(O2)11]-SP ineffective. Shallow concentration-response curves, variable efficacies and inhibition by atropine and mepyramine suggest that other mechanisms may influence contractile responses. Responses to [Lys5, MeLeu9, Nle10]-NKA(4-10) were inhibited competitively by MDL 29913 and MEN 10207 (pA2 values: 6.4 and 5.3, respectively). Antagonism by SR 48968 and GR 94800 was noncompetitive (both pK8 values 8.9). In summary, NK-2-preferring ligands showed superior potency as both binding competitors and contractile agonists, demonstrating that NK-2 receptors mediate detrusor muscle contraction, similar to the human detrusor. Tachykinins may play important roles in the micturition reflex and in regulating detrusor muscle blood flow in the dog.

Yohimbine prevents the retrograde flow of spermatozoa into the urinary bladder of dogs induced by xylazine

This study was carried out to determine whether yohimbine antagonizes the retrograde flow of spermatozoa into the urinary bladder of dogs caused by xylazine. Adult dogs were assigned to one of four groups of six dogs each and treated as follows: saline control, xylazine (2.2 mg/kg, i.m.), yohimbine (0.2 mg/kg, i.m.), yohimbine/xylazine (yohimbine, 0.2 mg/kg, i.m., followed 10 min later by xylazine, 2.2 mg/kg, i.m.). Pre- and post-treatment urine were collected by cystocentesis from all dogs. The mean (+/- SD) adjusted total number of spermatozoa in the post-treatment urine of xylazine-treated dogs (141.02 +/- 136.75 x 10(6)) was 15 times higher (P < 0.05) than the number in the post-treatment urine of control dogs (9.16 +/- 20.26 x 10(6), 1763 times higher (P < 0.05) than the number in the urine of yohimbine-treated dogs (0.08 +/- 0.20 x 10(6), and 56 times higher (P < 0.05) than the total number in the post-treatment urine of yohimbine/xylazine-treated dogs (2.54 +/- 4.54 x 10(6)). These results confirm that xylazine induces a significant (P = 0.007) displacement of spermatozoa into the urinary bladder of dogs and demonstrate that pre-treatment with yohimbine prevents this effect.

Subtypes of purinoceptors in rat and dog urinary bladder smooth muscles

1. Both adenosine and adenosine 5'-triphosphate (ATP) (10 microM and 100 microM) relaxed 10 microM acetylcholine (ACh)-induced contraction of rat bladder strips, which was completely antagonized by 100 microM 8-(p-sulphophenyl) theophylline. In dog bladder neither adenosine nor ATP inhibited ACh-induced contraction. 2. P2x-purinoceptor agonists contracted both rat and dog bladder strips with the potency order of alpha,beta-MeATP > ATP > ADP. 3. Alpha,beta-MeADP (100 microM) induced a contraction of the rat bladder strip even after desensitization of P2x-purinoceptors but failed to contract the dog bladder strip. 4. 2-MeSATP (1 microM to 300 microM) concentration-dependently induced contraction of rat bladder strips; this contraction was significantly inhibited after desensitization of P2x-purinoceptors. Cibacron blue 3GA (100 microM) antagonized the drug at concentrations lower than 30 microM, whereas it augmented the response to the drug at concentrations above 30 microM. 5. ADP beta S (1 microM to 1 mM) concentration-dependently induced contraction of rat bladder strips after desensitization of P2x-purinoceptors; a contraction which was significantly antagonized by cibacron blue 3GA (100 microM). 6. It is concluded that three subtypes of purinoceptors, P1 (mediating relaxation), and P2x and another type of P2 (mediating contraction), exist in rat urinary bladder smooth muscle, whereas a single subtype of the receptor, P2x-purinoceptor (mediating contraction) occurs in dog urinary bladder smooth muscle.

A study on the viscoelastic properties of the urinary bladder in dogs.

This experimental study was designed and carried out in order to investigate the participation of the collagen and muscular tissues on the viscoelastic properties of the bladder wall. Sixty-five adult male mongrel dogs were utilized. These animals were divided into 5 groups: control group (n = 10); dogs (n = 10) receiving 2.5 mg/kg atropine (cholinergic antagonist); dogs (n = 10) receiving 0.7 mg/kg verapamil (calcium extracellular inflow blocker); dogs (n = 10) receiving 0.1 mg/kg/min nitroprusside (intracellular calcium blocker), and dogs (n = 25) receiving EGTA (a calcium-chelating agent) at increasing doses from 90 to 450 mg/kg. Based on a mathematical model, we have demonstrated that: (1) the collagen component is responsible for the elastic properties; (2) the muscle component is responsible for the viscoelastic properties; (3) the viscoelastic properties have an active element which is affected by calcium total depletion, and (4) such viscoelastic properties are not dependent on cholinergic stimulation.

Acetylation of 2-aminofluorene derivatives by dog hepatic microsomes

Dog urinary bladder is a target organ of carcinogenic arylamines. However, dog hepatic and urothelial cytosols lack acetylation enzymes that are capable of activating N-hydroxy metabolites of arylamines, suggesting that other enzymes may be involved. In the present study, we found that dog liver microsomes were capable of N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and that these activities were inhibited by paraoxon. The 0.25% Triton X-100 extractable fraction of microsomes was resolved on an ion-exchange column into three different proteins that retained these activities. Two of these proteins, designated as enzyme I and enzyme II, were further chromatographed on a Sephacryl S-300 column. As judged from the gel filtration profile, the mol. wt of enzyme I was approximately 180 kDa and that of enzyme II was greater than 700 kDa. SDS-PAGE analysis showed that the subunit weight of enzyme II was approximately 150 kDa. In addition to N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-OH-AAF, these three enzymes were capable of the deacetylation of 2-acetylaminofluorene, N-OH-AAF and 4-nitrophenyl acetate. The ability of these microsomal enzymes to activate N-hydroxylated aromatic amines and the presence of these enzymes in urothelial cells, reported previously, suggests that they may play an etiological role in the carcinogenicity of these agents in the dog.

Retrograde flow of spermatozoa into the urinary bladder of dogs during ejaculation or after sedation with xylazine

SUMMARY Retrograde flow of spermatozoa into the urinary bladder of dogs during ejaculation or after administration of xylazine was examined. In experiment 1, the mean (± SD) spermatozoal concentration in urine collected by cystocentesis before ejaculation was 0.322 ± 0.645 × 106/ml. After ejaculation, motile spermatozoa were present in the urine collected by cystocentesis from 12 of 15 dogs, and the concentration of spermatozoa in the urine (5.139 ± 7.014 × 106/ml) was higher (P &lt; 0.025) than the concentration in the urine collected before ejaculation. The percentage of the total number of spermatozoa that were displaced during ejaculation and flowed into the urinary bladder (retrograde flow) ranged from 0 to 99.75% (24.67 ± 33.98%). In experiments 2 and 3, administration of xylazine to sexually rested dogs induced retrograde flow of spermatozoa into the urinary bladder. In experiment 2, all dogs had spermatozoa in urine collected after xylazine administration, with motile spermatozoa present in the urine from 9 of 10 dogs. In experiment 3, urine collected from dogs before administration of xylazine was azoospermic or contained few, nonmotile spermatozoa (0.063 ± 0.135 × 106/ml), whereas urine collected after administration of xylazine had more (P &lt; 0.025) and motile spermatozoa (3.717 ± 4.273 × 106/ml). In experiment 4, administration of xylazine to dogs after ejaculation did not increase the concentration of spermatozoa in the urine. Results indicate that spermatozoa flow into the urinary bladder of dogs during ejaculation or after administration of xylazine to sexually rested dogs.

Urinary bladder blood flow

The blood flow of the dog urinary bladder measured by radioactive microsphere technique was compared to the clearance of locally injected 99mTechnetium pertechnate (99mTc) in the bladder wall. In semilogarithmic plots the 99mTc washout curves showed a multiexponential course. From the initial slopes (median 5.7 min) the bladder blood flow was calculated to be only 30-62% of the results obtained from the radioactive microsphere technique (blood flow in the muscular layer 21.7-44.8 ml/100 g/min). These lower values imply that the rate of removal of the hydrophilic tracer 99mTc at these flow rates is limited by the capillary diffusion capacity. The multiexponential curves reflect recirculation and excretion of 99mTc by the kidneys with accumulation of 99mTc in the bladder. It is concluded, that clearance studies of locally injected 99mTc in the bladder wall are unable to evaluate bladder blood flow.

Central Actions of Bethanechol on the Urinary Bladder in Dogs

Bethanechol has been used to promote bladder emptying although the clinical efficacy of bethanechol remains uncertain. Both laboratory and clinical observations suggest that the pharmacologic actions of bethanechol are dependent to some extent on intact reflex mechanisms of the bladder. The objective of this study was to determine the effects of central nervous system administration of bethanechol on reflex detrusor contractions. Twelve isolated cross perfusion studies of the central nervous system in dogs were done to determine the cystometric response to central bethanechol administration. Central bethanechol resulted in reflex bladder contractions, decreased cystometric volume, increased maximum intravesical pressure, and increased periurethral EMG activity. This study demonstrated a central nervous system site of pharmacologic activity of bethanechol.

The absorption of trichloroethylene and its metabolites from the urinary bladder of anesthetized dogs

In order to examine the absorption of trichloroethylene (TRI) and its metabolites from the urinary bladder of dogs, we injected TRI and its metabolites, i.e., chloral hydrate (CH), free trichloroethanol (F-TCE), trichloroacetic acid (TCA) and conjugated trichloroethanol (Conj-TCE), into the urinary bladder of anesthetized dogs, and measured the agents and their respective metabolites in the blood or serum, urine and bile. The percentage of water absorbed from the urinary bladder was 10–20% 2 h after the administration of all substances. The percentage of agents absorbed was 60–70% for the TRI and TCA groups, and 50–60% for the CH, F-TCE and Conj-TCE groups 2 h after administration. The combined urinary and biliary excretion rates of the absorbed materials from the urinary bladder 2 h after administration were 46% for F-TCE, 30% for CH, 6% for Conj-TCE and 0.5–1.0% for TRI and TCA. Urinary re-excretion rates of the total excreted amounts were 65–70% in TRI, CH and F-TCE groups, about 50% in TCA and 99% in Conj-TCE group. It is possible that all of the substances administered, particularly F-TCE, are metabolized to Conj-TCE in the urinary bladder.

Scanning electron microscopy of changes in the urinary bladder in dogs treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN).

The fine structures of the bladder mucosa during BBN carcinogenesis and of bladder tumors induced by BBN in dogs were examined by scanning electron microscopy. Normal dog bladder mucosa was covered with polygonal superficial cells with peaked microridges, but no microvilli. Microridges and uniform microvilli were observed in hyperplastic mucosa. In low grade papillary tumors induced by low doses of BBN, pleomorphic microvilli predominated. In dogs which received high doses of BBN, bizarre pleomorphic microvilli and blebs were observed in papillary lesions, whereas bumpy surfaces with thick short microvilli were observed in non-papillary lesions. These sequential changes which were observed in dog bladder mucosa during BBN carcinogenesis parallelled the changes that occurred in the process of chemical carcinogenesis in rodents, and dog bladder tumors were similar to those of rodents and of human bladder cancers.

イヌおよびヒトぼうこう条片に対するプロスタグランジン（ＰＧｓ）の作用

イヌおよびヒトぼうこう条片に対するプロスタグランジン（ＰＧｓ）の作用

The actions of kinin antagonists on B 1 and B 2 receptor systems

The newly discovered bradykinin antagonists [Thi 5,8, D-Phe 7]Bradykinin, supplied by J.-M. Stewart and three other compounds, [D-Phe 7]BK, [Thi 5,8,D-Phe 7]Bradykinin and [Thi 6,9,D-Phe 8]Kallidin synthesized in our laboratory, were tested for their ability to antagonize bradykinin in four B 2 receptor systems, the guinea-pig ileum, the rabbit jugular vein, the dog carotid artery and the dog urinary bladder as well as against desArg 9-bradykinin in the rabbit aorta (a B 1 receptor system). [D-Phe 7]Bradykinin is a partial agonist, while [Thi 5,8, D-Phe 7]Bradykinin and [Thi 6,9,D-Phe 8]Kallidin are pure antagonists, the second one showing little BK-like activity on three of the four preparations. The kallidin analogue is more potent in all preparations than the bradykinin one. The two [Thi 5,8,D-Phe 7]BK (that supplied by J.-M. Stewart and that prepared in our laboratory) show very similar affinities in all preparations. The bradykinin analogue as well as the kallidin one are also active against desArg 9-bradykinin in the rabbit aorta, at concentrations similar to those active on B 2 receptor systems. The kinin antagonists are however specific for the kinins, since they do not interfere with the myotropic effects of angiotensin or substance P (SP) in the various preparations.

DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo.

The presence of relatively high levels of prostaglandin H synthase (PHS) in the dog urinary bladder and its ability to mediate the activation of carcinogenic arylamines to DNA-bound products in vitro suggests the involvement of this enzyme in arylamine-induced bladder carcinogenesis. Since the PHS-dependent metabolism of 2-naphthylamine (2-NA) had been shown to yield both ring- and N-oxidation products in vitro, we compared the reactivity of 3H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene, and 2-amino-1-naphthol (2-AN) toward DNA and protein. In the PHS-incubation system, all three derivatives bound at high levels to protein, but only N-OH-2-NA and 2-AN bound appreciably to DNA. Though ring-oxidation has usually been considered a detoxification pathway, the covalent binding of [3H]2-AN to DNA was found to occur readily under aerobic conditions and was enhanced at acidic pH. At pH 5 in air, the reactivity of [3H]2-AN with nucleic acids and protein was in the order: serum albumin greater than tRNA greater than poly G greater than poly C greater than DNA greater than poly A greater than rRNA greater than poly U. Enzymatic hydrolysis of DNA reacted with [3H]2-AN and subsequent analysis by h.p.l.c. indicated the presence of several carcinogen-nucleoside adducts. The major product was characterized as N4-(deoxyguanosin-N2-yl)-2-amino-1,4-naphthoquinoneimine; and two minor products were tentatively identified as N4-(deoxyadenosin-N6-yl)-2-amino-1,4-naphthoquinoneimine and a deoxyguanosin-N2-yl adduct of a naphthoquinoneimine dimer. These adducts accounted for approximately 60% of the total DNA binding obtained by incubation of [3H]2-NA with PHS in vitro and for approximately 20% of the [3H]2-NA bound to dog urothelial DNA in vivo. The remaining adducts were identical to those previously reported as products of the reaction of N-OH-2-NA with DNA. These results suggest that a minor proportion of the DNA adducts found in vivo may be formed by PHS-activation of 2-NA in the target tissue. Furthermore, the reactivity of 2-AN with cellular nucleophiles, presumably through formation of 2-imino-1-naphthoquinone or a protonated 4-naphthocarbenium ion, indicates that ring-oxidation products of arylamines and of other carcinogenic aryl compounds should be evaluated as proximate carcinogenic metabolites.

Rapid induction of carcinoma in situ in dog urinary bladder by sequential treatment with N-methyl-N'-nitrosourea and N-butyl-N-(4-hydroxybutyl)-nitrosamine.

A highly atypical intraepithelial proliferation, which was interpreted as carcinoma in situ, developed in the urinary bladder of 2 beagle dogs after repeated intravesical instillations of N-methyl-N'-nitrosourea followed by oral administration of N-butyl-N-(4-hydroxybutyl)nitrosamine. The latent period was 37 weeks. The technique may provide a useful model to study the natural history of bladder cancer, particularly carcinoma in situ.