Modulation of mitomycin C mutagenicity on Saccharomyces cerevisiae by glutathione, cytochrome P-450, and mitochondria interactions

Mutation Research 390 Ž1997. 113–120

Modulation of mitomycin C mutagenicity on Saccharomyces cereÕisiae by glutathione, cytochrome P-450, and mitochondria interactions Carlo Rossi a

a,)

, Paola Poli a , Alessia Candi a , Annamaria Buschini

b

Istituto di Genetica, UniÕersita` degli Studi di Parma, Viale delle Scienze, 43100 Parma, Italy b Biotecnologika scrl., Õia Bertani 6, 43035 Felino (PR), Italy Received 3 September 1996; revised 5 December 1996; accepted 6 December 1996

Abstract It is well established that most anticancer drugs also have mutagenic effects and require metabolic activation before exerting their mutagenicrantiblastic activity. Antitumoral compound effects strongly depend on the biochemicalrphysiological conditions of the tumoral cells, and especially on the activation of specific drugs metabolizing enzymes and on respiration. We examined the mitomycin C-induced mutagenic effects on the D7 strain of Saccharomyces cereÕisiae and on its derivative mitochondrial mutant r8 at different contents of glutathione and cytochrome P-450, molecules able to activaterdetoxicate xenobiotics. The mutagenic activity of the drug was evaluated as frequency of mitotic gene conversion and reversion in different physiological conditions. The highest frequencies of reversion and especially of gene conversion were observed at the highest cytochrome P-450 contents in the D7 strain with a further increase at high glutathione level. In the respiratory-deficient strain, the highest frequency of convertants was shown at low glutathione level and lack of cytochrome P-450. These results suggest the relevance of mitochondrial functionality for the expression of genotoxic activity of this anticancer drug. Keywords: Antiblastic drug; Yeast; Respiratory deficient mutant; Short-term mutagenesis assay; Gene conversion; Point mutation

1. Introduction Mitomycin C ŽMMC. is a quinone-containing natural antibiotic used in clinical cancer chemotherapy against a variety of solid neoplasms Žbreast, prostate, bladder, colorectal, gastric and lung cancers.. How-

) Corresponding author. Tel.: q39 Ž521. 905-608; Fax: q39 Ž521. 905-604; E-mail: [email protected]

ever, emergence of drug-resistant tumor cells limits the clinical effectiveness of MMC. Mitomycin C requires enzymatic activation for its cytotoxic activity w1–4x. Following bio-activation, MMC is capable of producing oxyradicals as well as DNA alkylating species due to the presence of both quinone and aziridine moieties, respectively w3,5–7x. The active oxygen species can produce DNA strand breaks w8x, and this activity as well as the cross-linking of DNA have been implicated in the biological action of MMC w1,3,5–12x.

1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 0 7 - 4

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C. Rossi et al.r Mutation Research 390 (1997) 113–120

Multiple drug resistance mechanisms are present in resistant cells, including decreased drug activation, increased drug detoxication, and decreased accessibility of DNA targets. In particular, MMC cellular resistance can be modulated by an increased intracellular level of glutathione ŽGSH. w10,13–18x. Cytochrome P-450 is a product of a multigene family, and catalyzes the activation and the detoxication of a wide variety of exogenous as well as endogenous compounds. In general, cancer cells express lower amounts of cytochrome P-450 as compared to normal cells w19–22x. Drug activity can also be dependent on mitochondrial functionality as observed by the different behavior of oxygenic and hypoxic tumor cells versus antitumor chemicals w19,23–25x. However, MMC does not inhibit mitochondrial respiration in isolated rat hepatocytes w26x. In any case, MMC has been shown to be preferentially toxic to hypoxic tumor cells both in vitro w5,27x and in vivo w28x. There is clear evidence that the cytotoxicity and genotoxicity of antiblastic compounds are governed by the different biochemicalrphysiological conditions of the tumoral cells, determined especially by the activation of specific drug-metabolizing enzymes and respiration. We examined the mutagenic effects induced by MMC in two strains, D7 w29x and D7r8 Žrespiratorydeficient strain, obtained in our laboratory., of Saccharomyces cereÕisiae in different conditions of expression of glutathione and cytochrome P-450 contents. S. cereÕisiae may provide a valuable model for understanding factors governing the movement of a variety of drugs w30–32x. A previous study was performed to choose the experimental conditions for cellular concentration of glutathione and cytochrome P-450 Žunpublished data..

2. Materials and methods 2.1. Saccharomyces cereÕisiae strains The strain D7 was used to determine the frequencies of mitotic gene conversion of the trp-5 locus and reversion of ilÕ1-92 mutant. D7r8 strain is a D7 ‘petite’ mutant lacking mito-

chondrial DNA, obtained after ethidium bromide treatment w33x. The two strains were characterized for convertant and revertant spontaneous frequencies in different experimental conditions. Cellular cultures in yeast extract ŽYE., 0.2 or 20% glucose, wro 10y2 M, L-buthionine sulfoximine maintained for 16–18 h in an alternative shaker Ž120 rpm. at 288C, were harvested during the logarithmic phase of growth. After centrifugation, the cells were resuspended, at 5 = 10 7 cellsrml, in phosphate-buffered YE, pH 7, with the same glucose and L-buthionine sulfoximine concentration of growth conditions. The samples were maintained for 2 h at 288C in an alternative shaker. The cells were washed twice, resuspended in distilled water, and plated on a medium without tryptophan, about 2 = 10 6 cells per plate, on a isoleucinefree medium, about 2 = 10 7 cells per plate, and finally on a complete medium, about 200 cells per plate. Furthermore, the convertant and revertant frequencies induced by hycanthone and ethyl methanesulfonate on the two strains were assessed in cells during the stationary growth phase w29x. After the treatment, the cells were plated on a medium without tryptophan, about 2 = 10 6 cells per plate, on an isoleucine-free medium, about 2 = 10 7 cells per plate, and finally on a complete medium, about 200 cells per plate. In these strains, GSH and cytochrome P-450 cellular concentrations were determined before their utilisation in mutagenesis assays. 2.2. Cell extract and GSH assay Cellular cultures in YE, 0.2 or 20% glucose, wro 10y2 M, L-buthionine sulfoximine maintained for 16–18 h in an alternative shaker Ž120 rpm. at 288C, were harvested during the logarithmic phase of growth. Cells were washed twice with 0.9% NaCl and resuspended in distilled water. The cell suspension was subjected to heat shock Ž1008C, 90 s. and centrifuged at 6000 rpm for 15 min at 48C. The GSH concentration in the supernatant fraction was determined immediately using a modified Morineau w34x method. Fifty microliters of filtered sample was incubated for 2 min with 50 ml of 80 mM o-phthalaldehyde and 50 ml of a solution of 0.3 M sodium acetate in 8% acetonitrile, pH 7.4 Ži.e., mobile phase.

C. Rossi et al.r Mutation Research 390 (1997) 113–120

and injected in Water M510 HPLC ŽmBondapack C18 column. equipped with a M420-AC fluorescence detector and a Water 746 data module. 2.3. Cell extract and cytochrome P-450 assay Cellular cultures in YE, 0.2 or 20% glucose, wro 10y2 M L-buthionine sulfoximine, maintained for 16–18 h in an alternative shaker Ž120 rpm. at 288C, were harvested during the logarithmic phase of growth. Cells were washed twice with a buffer containing 50 mM Tris HCl, 10 mM EDTA, 0.8 M sorbitol, pH 7.4 and resuspended in the same buffer at a final cellular concentration of 10 9 cellsrml. Cytochrome P-450 was determined by a modified Omura and Sato w35x method. Yeast suspension was placed in each of two spectrophotometer cuvettes and reduced by the addition of a few grains of sodium dithionite. The baseline between 400 and 500 was then recorded using a Cary spectrophotometer. Carbon monoxide was then bubbled through the test cuvette and the scan repeated. The peak height at 450 nm above the baseline was then used to calculate the concentration of cytochrome P-450, assuming an extinction coefficient of 91 cmy1 mMy1 . 2.4. Mutagenesis assays Cellular cultures in YE, 0.2 or 20% glucose, wro 10y2 M L-buthionine sulfoximine, maintained for 16–18 h in an alternative shaker Ž120 rpm. at 288C, were harvested during the logarithmic phase of growth. After centrifugation, the cells were resuspended, at 5 = 10 7 cellsrml, in phosphate-buffered YE, pH 7, with the same glucose and L-buthionine sulfoximine concentration of growth conditions, at determined doses of MMC. The samples were maintained for 2 h at 288C in an alternative shaker. The cells were washed twice, resuspended in distilled water, and plated at the scheduled concentration on solid complete medium 2% glucose, Ž; 200 cellsrplate. to determine survival titer, and on selective mineral medium w36x added of adenine and isoleucine Ž; 2 = 10 6 cellsrplate. or adenine and tryptophan Ž; 2 = 10 7 cellsrplate. to detect gene conversion and reversion frequencies, respectively. All experiments were performed at least three times and data compared for reproducibility. Pooled

115

data are presented. The data were analyzed using the 2-fold rule w37x in which a response is considered positive if the average response for at least two consecutive dose levels was more than twice the spontaneous frequencies. The data obtained were also subjected to the Student’s t-test.

3. Results 3.1. Saccharomyces cereÕisiae strains The gene conversion and point mutation spontaneous frequencies of the D7 strain and its r8 derivative induced by hycanthone and ethyl methanesulfonate on the two strains were assessed in cells during stationary growth phase ŽTable 1.. The petite strain shows a very different behavior to the parental strain when mutagenicity is induced by hycanthone, while the effects appear to be induced in a more similar way on the two strains by ethyl methanesulfonate. The gene conversion and point mutation spontaneous frequencies of the two strains were also determined under different experimental conditions ŽTable 2.. The two strains do not show significant differences among various experimental conditions Ž0.2 or 20% glucose wro 10y2 M L-buthionine sulfoximine. both for convertant and revertant frequencies. The petite strain shows a larger variance than the D7 strain for gene conversion induction and the same behavior of the parental strain for point mutation induction. 3.2. Cytochrome P-450 and GSH cellular contents The cytochrome P-450 and GSH cellular contents of the D7 and D7r8 strains were determined in logarithmic growth phase cells ŽTable 3.. Cytochrome P-450 induction appears strongly dependent on glucose concentration w38–40x and at low glucose concentration Ž0.2%. cytochrome P-450 was not detectable in both strains. Furthermore, the P-450 content is not considerably affected by complete loss of mitochondrial functionality both at high and low glucose concentrations and this suggests that respiratory deficiency condition on its own is not sufficient to induce cytochrome P-450 high expression.

C. Rossi et al.r Mutation Research 390 (1997) 113–120

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Table 1 Induction of convertants and revertants in the D7 and D7r8 strains by ethyl methanesulfonate ŽEMS. and hycanthone ŽHYC. Treatmentrdose

EMS ŽmM. 0 25 50 100 150 HYC Žmgrml. 0 10 20 40 80 100

titer Ž%. D7

D7r8

100.0 Ž5 391.

100.0 Ž5 868. 98.9 Ž5 803. 86.3 Ž5 064. 67.9 Ž3 984. 20.6 Ž1 209.

90.3 Ž4 869. 66.9 Ž3 555. 28.3 Ž1 503. 100.0 Ž5 771. 91.0 Ž5 252. 98.7 Ž5 696. 86.8 Ž5 009. 78.5 Ž4 530. 21.2 Ž1 223.

100.0 Ž5 432. 87.4 Ž4 748. 91.7 Ž4 981. 85.3 Ž4 633.

Locus trp5 convertantsr10 5 survivors

Locus ilÕ1 revertantsr10 6 survivors

D7

D7

D7r8

0.56 Ž303. 15.34 Ž747. 66.47 Ž2 363. 131.27 Ž1 973. 0.94 Ž495. 1.13 Ž593. 1.32 Ž752. 1.72 Ž862. 2.27 Ž1 028. 5.79 Ž708.

a a a

1.27 Ž745. 10.20 Ž592. 31.34 Ž1 587. 71.79 Ž2 860. 162.37 Ž1 963. 1.18 Ž641. 32.13 Ž1 515. 56.19 Ž2 799. 98.58 Ž4 567.

0.45

D7r8 Ž240.

a a a a

a a a

109.61 Ž5 337. 357.30 Ž12 702. 449.50 Ž6 756. 0.31 0.35 0.36 0.40 0.76 2.45

b b b

0.41 Ž241. 15.92 Ž924. 50.65 Ž2 565. 154.17 Ž6 142. 488.34 Ž5 904.

Ž179. Ž184. Ž205. Ž200. Ž344. Ž300.

b b b b

0.49 Ž266. 2.15 Ž1 021. 3.34 Ž1 664. 5.75 Ž2 664.

Stationary phase cells, glucose concentration 2%. Number in brackets, actual colony counts. Values represent pooled data of three independent experiments. a,b Cell suspensions were plated: a 2 = 10 5 cellsrplate; b 2 = 10 6 cellsrplate.

In both strains, L-buthionine sufoximine decreases the intracellular GSH level ŽTable 3., also if a larger decrease is shown at 20% glucose Ž70 and 55% for the D7 and the D7r8 strain, respectively. in comparison with 0.2% glucose concentration Ž38% for the D7 and 28% for the D7r8.. It is also evident at a higher level of GSH in the petite strain. 3.3. Mutagenesis assays

3.3.1. Point mutation The reversion frequency in the D7 strain is weakly increased at low glucose concentration without any significant effect by GSH level. On the contrary, a simultaneous high content of P-450 and GSH induces a larger amount of ilÕ-revertants. For the same mutagenic effect, the D7r8 strain shows more sensitivity at low GSH content, especially under P-450 lack.

The results of mutagenesis assays performed on logarithmic growth phase cells in different culture and incubation conditions Ž0.2 and 20% glucose; wro L-buthionine sulfoximine. are reported in Table 4 for the D7 strain and the petite mutant.

3.3.2. Gene conÕersion The convertant frequency appears strongly increased under all cultural conditions for both strains. The D7 strain shows a higher frequency on P-450 induction conditions than at low glucose concentra-

Table 2 Spontaneous frequencies of gene conversion and point mutation in the D7 and D7r8 strains at different concentrations of glucose with Žq. or without Žy. L-buthionine sulfoximine ŽBSO. 10y2 M Glucose

Locus trp5 convertantsr10 5 survivors

Locus ilÕ1 revertantsr10 6 survivors

Ž%.

D7

D7r8

D7

D7r8

0.2 yBSO 0.2 qBSO 20.0 yBSO 20.0 qBSO

0.90 " 0.13 0.98 " 0.31 0.89 " 0.16 1.27 " 0.24

1.34 " 0.61 1.62 " 0.57 1.45 " 0.56 1.55 " 0.42

0.51 " 0.27 0.50 " 0.13 0.46 " 0.21 0.48 " 0.20

0.46 " 0.12 0.41 " 0.14 0.45 " 0.22 0.45 " 0.16

Cells were harvested during the logarithmic phase of growth. Values represent mean " SD of 5 independent experiments.

C. Rossi et al.r Mutation Research 390 (1997) 113–120 Table 3 Cytochrome P-450 and glutathione ŽGSH. contents in the D7 and D7r8 strains at different concentrations of glucose with Žq. or without Žy. L-buthionine sulfoximine ŽBSO. 10y2 M

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lack is correlated to increasing MMC efficacy; besides, glutathione appears to have a weak protective effect, at low glucose concentrations.

Glucose BSO 10y2 M P-450 GSH Ž%. Žpmolr10 8 cells. Žmgrg d.wt.. D7

D7r8

0.2 0.2 20 20

y q y q

ND ND 23.1"1.2 21.9"1.1

2.12"0.11 1.31"0.09 2.77"0.08 0.83"0.10

0.2 0.2 20 20

y q y q

ND ND 18.6"0.9 16.3"1.1

2.69"0.08 1.94"0.09 3.22"0.10 1.46"0.11

Values represent mean"SD of at least 3 independent experiments. ND, not detectable.

tion. The partial inhibition of glutathione synthesis by L-buthionine sulfoximine produces a weak increase of MMC mutagenic potency at 0.2% glucose concentration, whereas at 20% glucose concentration, i.e., high P-450 content, glutathione inhibition reduces the MMC effects. The respiratory-deficient strain shows a different behavior: in particular, P-450

4. Discussion It is well established that antiblastic drug-induced cytotoxicity andror genotoxicity are determined by various mechanisms of interaction with biological macromolecules. Mitomycin C mainly acts as a bifunctional DNA-alkylating agent, even if DNA damage induced by MMC-produced hydroxyl radical and other reactive species of oxygen are known. However this antitumor substance was shown only to be active when in its reduced form. Moreover the role of different biochemical and physiological cellular conditions, such as inductionrinhibition of cytochrome P-450, glutathione and respiration, must be taken into account to understand the chemotherapeutic compound mechanisms. For this purpose, gene conversion and point mutation induction by MMC were evaluated in two S. cereÕisiae strains, the D7 strain and its petite mutant

Table 4 Induction of convertants and revertants in the D7 and D7r8 strains by mitomycin C at different glucose concentrations with Žq. or without Žy. L-buthionine sulfoximine ŽBSO. 10y2 M Glucose

Dose

Titer Ž%.

Ž%.

Locus trp5 convertantsr10 5 survivors

Locus ilÕ1 revertantsr10 6 survivors

Žmgrml.

D7

D7r8

D7

D7r8

D7

D7r8

0.2 yBSO

0 50 100 200

100 Ž5 034. 94 Ž4 752. 117 Ž5 906. 90 Ž4 528.

100 Ž4 770. 99 Ž4 722. 104 Ž4 950. 95 Ž4 510.

0.86 Ž432. 4.74 Ž2 252. 5.5 7.14 Ž4 216. 8.3 12.70 Ž5 750. 14.8

2.15 Ž1 026. 12.75 Ž6 021. 5.9 19.20 Ž9 504. 8.9 45.75 Ž20 670. 21.3

0.18 Ž90. 0.25 Ž118. 1.4 0.35 Ž206. 1.9 0.44 Ž200. 2.4

0.60 Ž285. 0.65 Ž306. 1.1 0.70 Ž348. 1.2 1.19 Ž537. 2.0

0.2 qBSO

0 50 100 200

100 Ž4 598. 120 Ž5 510. 72 Ž3 302. 92 Ž4 256.

100 Ž5 447. 70 Ž3 786. 74 Ž4 047. 70 Ž3 786.

1.23 Ž566. 5.80 Ž3 196. 4.7 14.72 Ž4 860. 12.0 19.64 Ž8 358. 16.0

1.55 Ž843. 13.80 Ž5 226. 8.9 23.30 Ž9 429. 15.0 43.15 Ž16 338. 27.8

0.48 Ž220. 0.66 Ž364. 1.4 1.06 Ž350. 2.2 1.14 Ž486. 2.4

0.30 Ž163. 0.69 Ž261. 2.3 0.90 Ž364. 3.0 1.62 Ž613. 5.4

20.0 yBSO

0 50 100 200

100 Ž4 120. 106 Ž4 360. 76 Ž3 124. 58 Ž2 386.

100 Ž5 085. 110 Ž5 604. 81 Ž4 113. 82 Ž4 185.

0.90 Ž370. 11.94 Ž5 206. 13.3 16.83 Ž5 258. 18.7 37.46 Ž8 938. 41.6

2.05 6.10 11.85 22.35

0.20 Ž82. 0.36 Ž156. 1.8 0.49 Ž154. 2.5 1.04 Ž248. 5.2

0.62 Ž315. 0.57 Ž319. 0.9 0.63 Ž259. 1.0 0.71 Ž297. 1.1

20.0 qBSO

0 50 100 200

100 Ž4 788. 90 Ž4 320. 84 Ž4 029. 61 Ž2 940.

100 Ž4 674. 116 Ž5 433. 109 Ž5 109. 68 Ž3 177.

1.48 Ž708. 12.14 Ž5 244. 8.7 18.43 Ž7 425. 12.5 30.42 Ž8 943. 20.6

1.90 Ž888. 6.45 Ž3 504. 3.4 8.80 Ž4 497. 4.6 26.30 Ž8 355. 13.8

0.42 Ž201. 0.60 Ž258. 1.4 0.62 Ž249. 1.5 1.43 Ž420. 3.4

0.38 Ž177. 0.44 Ž240. 1.2 0.68 Ž348. 1.8 1.09 Ž345. 2.9

Ž1 041. Ž3 417. 3.0 Ž4 875. 5.8 Ž9 354. 11.9

Total number of counted colonies is given in brackets; values in italics represent the ratio between induced and spontaneous mutants.

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C. Rossi et al.r Mutation Research 390 (1997) 113–120

derivative D7r8 under different conditions of glutathione and cytochrome P-450 cellular contents. The mitochondrial functionality appears to affect the cytosolic GSH level ŽTable 3.. Many relationships are present between P-450 complex and mitochondria w41–44x. P-450 cellular content was shown to be dependent on glucose concentration, in accordance with literature data w38–40x. Besides, the level of P-450 in the r8 strain appears to be also affected by glucose concentration: loss of mitochondrial function does not result sufficient for P-450 induction ŽTable 3.. This suggests that the synthesis of P-450 in S. cereÕisiae could be caused by a catabolite of sugar, or an effector generated by this catabolite as demonstrated in another experimental framework w39x. The mutagenic effects assessed by point mutation and gene conversion induction ŽTable 4. were shown to be influenced by cellular conditions. The gene conversion appears to be a more sensitive target than reversion for MMC, possibly because of the more efficient alkylating action w6–8,12x rather than DNA damage by oxyradicals w8,45x. The induction of P-450 was shown to have different effects in the two strains considered which have a contradictory behavior when the cytochrome is present, especially for gene conversion. In particular, in the D7 strain, P-450 high levels are related to high mutation frequencies, whereas in the r8 strain, the highest mutation frequency was induced at low glucose concentration, i.e., P-450 lack. Our results confirm that the lackrpresence of cytochrome P-450 affects the MMC action, as also seen in a variety of cells w46x. The GSH level was shown to act in an alternative way on the two yeast strains, too. The D7 strain shows an increase in convertant induction at simultaneous higher GSH and P-450 contents and non-appreciable effects of glutathione at low glucose concentration. On the petite strain, the thiol has a detoxifying effect under condition where P-450 is lacking, and does not act when P-450 is induced. Contrasting results on the relationship between cellular MMC resistance and GSH cellular concentrations are known. In a large variety of cell lines w1,15– 17,47,48x, a GSH protective function was shown, eventually dependent of oxygenicrhypoxic conditions w49,50x, unlike other cell types w13,20,51–53x,

where relationships between drug sensitivity and low GSH levels were not found. Moreover, GSH was demonstrated to compete with DNA for the activated MMC w54x, forming non-toxic drug conjugates, and, at the same time, able to form a ternary MMC– GSH-DNA adduct. In addition, our results paradoxically suggest ŽTable 4. that the reductive metabolism of MMC, at the highest P-450 and GSH contents, leads, in the D7 strain, to the highest increase of DNA lesions, whereas, at the same conditions, it leads, in the r8 strain, to a detoxication, probably by formation of inactive metabolites. Respiratory function was shown to affect the MMC-induced genotoxicity, both through the oxyradical production and through the different cellular metabolism. As reported, MMC-resistant tumor cells contained a higher content of antioxidant enzymes w26x and the MMC sensitivity was increased in hypoxic conditions w27x. Our results confirm the relevance of mitochondrial functionality on mechanisms of drug action. A different mutagenic activity of MMC and a different way of activation were shown in the r8 strain with respect to the parental strain. At high P-450 and GSH contents, MMC shows the highest mutagenic activation in the respiratory sufficient strain, while the respiratory-deficient strain shows the lowest genotoxicity values. On the other hand, the lack of mitochondrial activity productrs in the r8 strain determines a MMC-increased sensitivity at low concentration of the two ‘activators’ cytochrome P-450 and GSH Ž0.2% glucose with L-buthionine sulfoximine.; this suggests that other MMC-activating factors might be ‘derepressed’ in respiratory-deficient conditions. Further investigations must be performed to understand the activationrdetoxication pathways in cells with a lack of mitochondrial activity. The yeast S. cereÕisiae seems to be a metabolic and genetic model able to clarify the action of xenobiotic by modulation of drug-metabolizing enzymes and mitochondrial functionality.

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