DNA damage in liver of rats treated with nitrofurantoin

Mutation Research, 105 (1982) 377-382

377

Elsevier Biomedical Press

D N A damage in liver of rats treated with nitrofurantoin* P a t r i z i a R u s s o , M a u r o P a l a , G u i d o N i c o l 8 , L e o n a r d o Santi a n d Silvio Parodi** Department o f Oncology (L.S., S.P.), Genoa University; Istituto Scientifico per 1o Studio e la Cura dei Tumori, Istituto Nazionale par la Ricerca sul Cancro (P.R., M.P., G.N.); 1-16132 Genoa (Italy)

(Accepted 22 June 1982)

Nitrofurantoin (NF), Furadantin, one of the most commonly used urinary-tract antiseptics, has been associated with acute and chronic pulmonary reactions and hepatic damage (Black et al., 1980; Sharp et al., 1980). Because m a n y other nitrofurans are carcinogenic in experimental animals (Cohen, 1978), NF has been assessed for potential rat carcinogenic activity. Published studies, even of negative results (Morris et al., 1969); Yahagi et al., 1974), are limited, as has been pointed out by other investigators. Moreover, NF, like known carcinogenic nitrofurans, is mutagenic and has DNA-modifying activity in bacterial systems (Yahagi et al., 1974, 1976) after addition of liver microsomal preparation. NF has also been tested for the induction of c h r o m o s o m e aberrations and of SCEs in the bone marrow of Chinese hamsters in vivo (Siebert et al., 1979). It induced only few c h r o m o s o m e aberrations and a marginally significant increase of SCEs which was not dosedependent. Wang et al. (1977) reported that the urine from humans treated with NF has mutagenic activity in S a l m o n e l l a t y p h i m u r i u m TA100. Significant protein covalent binding of 14C-NF occurs in vivo to kidney, liver and lung tissues in rats treated with i.v. doses. These data indicate that NF can be metabolized to reactive species that can alkylate tissues (Boyd et al., 1979). The present investigation reports the capacity of NF to induce damage in liver D N A of rats after treatment in vivo.

Materials and methods C h e m i c a l s . Nitrofurantoin (NF), trade name Furadantin, pharmaceutical grade, was purchased f r o m Formenti SpA, Milan (Italy); tetraethylammonium hydroxide

*This research was supported by Grants from Consiglio Nazionale delle Ricerche No. 81.01386.96, Special Research Project 'Control of Neoplastic Growth'. **To whom requests for reprints should be addressed. 0165-7992/82/0000-0000/$02.75 © Elsevier Biomedical Press

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was obtained f r o m E. Merck, Darmstadt (West Germany); 3,5-diaminobenzoic acid dihydrochloride was purchased f r o m Fluka AG, Buchs (Switzerland); carboxymethylcellulose was obtained from Sigma, St. Louis, (U.S.A.). All other chemical were reagent grade.

DNA damage in vivo/alkaline elution assay The assay, in vivo, for D N A damage was performed with 100-200 g S p r a g u e - D a w l e y male albino rats (CD Charles River). The c o m p o u n d examined was suspended in 0.9% NaCI with 1°7o of carboxymethylcellulose and administered by the i.p. route in 0.01 ml of vehicle/g b.w. Controls were injected i.p. with the same volume o f the same vehicle. Each group treated comprised at least 6 animals. At different times after treatment, rats were killed by cervical dislocation and exsanguinated. Livers were quickly removed and soaked in cold Merchant's solution (0.14 M NaCI, 1.47 mM KH2PO4, 2.7 m M KC1, 8.1 m M Na2HPO4, 0.53 m M Na2EDTA; p H 7.4). Livers were minced and then homogenized in a P o t t e r - E l v e h j e m homogenizer in 3 ml of Merchant's solution. After sedimentation of the large tissue fragments, nuclei and single cells remaining in the supernatant were centrifuged at 50 x g for 2 min and resuspended in 5 ml of Merchant's solution maintained at 4°C. Cells and nuclei (suspended in cold Merchant's solution) were counted in a hemocytometer. Alkaline elution was performed essentially according to Kohn et al. (1976), as previously described (Parodi et al., 1978), with minor modifications. About 106 cells were loaded on a Millipore (mixed esters of cellulose) filter (25 m m diameter, 5 #m pore size) and washed with cold Merchant's solution. The cells were lysed on the filter at r o o m temperature with 4.5 ml of 0.2% sodium lauroyl sarcosinate, 2 M NaC1, 20 mM Na2EDTA (pH 10), and the filter was washed with 2.5 ml of 20 m M Na2EDTA (pH 10). Single-stranded D N A was eluted in the dark with 13 ml of 60 m M tetraethylammonium hydroxide, 10 m M Na2EDTA (pH 12.3), at a p u m p speed of 0.2 m l / m i n , by collecting 13 ml after 60 min of elution. At the end of elution the D N A remaining on the filter was recovered by breaking up the filter in 2.6 ml of eluting solution. Eluted D N A and filter were assayed for D N A content by the following modification of the microfluorometric technique of Kissane and Robins (1958). To each 1-ml aliquot of the collected fraction, 0.1 ml of 100°70 (w/v) trichloroacetic acid was added. The samples were then refrigerated for several hours, and the precipitated D N A was obtained by centrifuging at 1750 × g for 30 min at 4°C. Supernatants were decanted, and 1 ml o f 80°/o ethanol was layered on each pellet. After a second centrifugation, the pellets were air dried for about 3 h at 40°C. 30/~1 of 40°70 (w/v) aqueous solution of 3,5-diaminobenzoic acid dihydrochloride were added to each sample, and the tubes were incubated for 30 min at 70°C. The tubes were cooled, then 1.5 ml o f 0.6 N perchloric acid were added to each tube. The fluorescence was read at 500 nm with an excitation wavelength of 400 nm in a P e r k i n - E l m e r model

379

3000 fluorimeter. Blank readings were made from tubes containing 1 ml o f eluting solution subjected to the same procedure. The recovery o f applied D N A was 80°7o or greater. The average elution rate constant is given by the following formula: K=

- I n (fraction o f D N A retained on filter) V

K is the average elution-rate constant ( m l - 1) o f D N A and V is the eluted volume in ml. As a first approximation, K is directly proportional to the number o f singlestrand breaks. Moreover, the above formula reflects the assumption of a first-order kinetics for D N A elution, always as a first approximation (Kohn et al., 1976). Tissue samples were taken, for histological examination, from at least 3 rats from each treatment schedule.

Results and discussion The LDs0 o f NF, after a single i.p. injection, was 112 m g / k g (Registry of Toxic Effects o f Chemical Substances, 1978). In Table 1 we report the median K values, the I and III quartile range, number o f experiments and statistical analysis o f results, for alkaline D N A fragmentation induced by NF in liver o f male rats. Table 1 and

TABLE

1

DNA FRAGMENTATION AFTER DIFFERENT

Dosage and time after treatment mg/kg

INDUCED

TREATMENT

BY N I T R O F U R A N T O I N

(FURADANTIN)

1N R A T L I V E R

SCHEDULES

Number of Expts.

Median K x

10 2

I-II1 quart range

Statistical a significance

h

28

72

6

1.76

1.62-2.11

N.S.

56

72

6

4.90

3.93-5.48

p < 0.002

112

72

11

6.20

5.10-8.07

p < 0.002

112

6

6

1.75

1.32-2.60

N.S.

112

24

8

2.52

1.83-2.89

N.S.

112

30

6

2.90

2.63-3.19

p = 0.02

112

48

9

6.13

4.00-7.04

p < 0.002

112

96

6

4.76

3.31-6.14

p< 0.002

112

168

6

2.07

1.62-3.31

N.S.

14

1.71

1.36-2.40

Cumulative controls

aprobability (two-tailed) that treated samples did not differ from their own control set, according to the non-parametric M a n n - W h i t n e y test (Siegel, 1956); N . S . = p > 0.1.

380

~-- 5 cq(D

4 3

2

.L.

I

0

0

l 28

I 56

{ 112

NF dosage (r~J/Kq) Fig. 1. Dependence of elution rate constant (K) on nitrofurantoin dosage. DNA from liver of Sprague-Dawley male rats, killed 72 h after treatment. Each point represents the median value of 6-11 Expts. Controls represent the median value of 14 Expts. Bars represent the I - l l l quartile range.

4 CNG

× 3 2

0

I I I 24 48 72 }k:~rs a f t e r t r e a t r 0 e n t

I 96

I 120

I 144

I 168

Fig. 2. Dependence of elution rate constant (K) on time after treatment. Nitrofurantoin dosage 112 m g / k g i.p., DNA from liver of Sprague-Dawley male rats. Each point represents the median value of 6-11 Expts. Controls represent the median value of 14 Expts. Bars represent the I - I I I quartile range.

381

Figs. 1 and 2 show dose and time dependence of D N A damage induced in liver after a single i.p. injection. The doses given were: 28, 56 and 112 m g / k g . Damage (for the range of doses tested) was maximal at 112 m g / k g . The relationship between amount of D N A damage and time, for a dosage of 112 m g / k g , is shown in Table 1 and Fig. 2. D N A damage was evident 30 h after treatment, remained high for 4 8 - 7 2 h, decreased at 96 h and fell to control levels 168 h after treatment. Our alkaline elution method was calibrated against X-rays (Brambilla et al., 1979). A K above controls of 0.1 corresponds to about 3.2 breaks/109 dalton. As a consequence, the highest levels of damage (112 m g / k g at 48 and 72 h) correspond to about 1.4 breaks/109 dalton. Light microscopy of liver histological preparations revealed a cloudy degeneration around the centrolobular vein. At 72 h rare isolated necrotic cells were sometimes also observed. As an average no relationship between seriousness of the lesions and D N A damage was established. The results of mutagenicity studied and the formation of adducts with macromolecules (Yahagi et al., 1974, 1976; Wang et al., 1977; Boyd et al., 1979; Siebert et al., 1979), and our present studies, all strongly support the view that the potential carcinogenicity of NF should be carefully re-examined, particularly in view of its widespread use in human urinary infections. At 1/4 LDs0 (28 m g / k g ) no significant effect was scored. It may be possible that up to that dose the animals can metabolize NF to non-genotoxic metabolites, whereas at higher doses, D N A interaction may occur. In other words a 'threshold p h e n o m e n o n ' cannot be excluded.

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