Roles of flavoproteins in degradation of mexacarbate in rats

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

10,

67-78

(1979)

Roles of Flavoproteins of Mexacarbate

in Degradation in Rats’

E. G. ESAAC AND F. MATSUMURA of

Department

Entomology.

Received

The nature of a reductive liver was investigated. The ments and its requirements result of purification reductive activity flavin cofactor water-soluble

was

of

University October

5, 1977:

system which actively system is characterized for a flavin cofactor

degrade

mexacarbate

first

22,

53706

1978

to 4-N-desmethylmexacarbate

and

and treatAs a

for the above flavoproteinthen

to polar

the activity even to a greater extent. Reduction of the sulfoxide group of the carbophenothion sulfoxide is reported by DeBaun and Menn (3) to require both NADPH and FAD for full reductive enzyme activity. With regard to the rat liver microsome enzyme system, NADPH is reported to be essential for reductive dechlorination or dehydrochlorination of toxaphene or its toxicants A and B (4). Khalifa et al. (4) suggested the involvement of cytochrome P-450 in these reductive enzyme reactions. It is interesting to note that the presence of a flavin cofactor is a common factor for these in vitro enzyme systerns. On the other hand, the liver is essentially an oxidative organ and the presence of such reductive degradation activities is rather puzzling. Also, it has never been made clear whether these reductive activities are due to several different systems or to general all-purpose system such as the P-450 involving process. The purpose of this study is twofold; first, to clarify the role of flavin cofactors in the reductive degradation of a pesticidal substrate, mexacarbate, and second, to identify the system which is responsible for such a degradation system.

INTRODUC’HON

and

March

Wisconsin

attempts the system responsible Under anerobic conditions such

Reductive enzyme reactions by microorganisms are known to play a major role in the degradation and eventual elimination of certain xenobiotics such as chlorinated hydrocarbon insecticides, from the environment. In higher animals, the nitroreductase is a known reductive enzyme system and is of considerable importance from the viewpoints of pharmacology and toxicology (1). Recently, other reductive systems have been reported which are actively involved in detoxication processes. Two enzyme systems from liver have been reported by other workers to exhibit reductive activity on pesticides; namely. the 12,000g supernatant (2, 3) and the microsomes (4). Hassall (2) reported that pigeon liver supernatant requires NADPH as a cofactor to convert DDT to TDE and that this enzyme activity is lost upon heating the enzyme preparation. On the other hand, addition of riboflavin to the heated (75°C 20 min) enzyme preparation restored

’ Supported by College of Agricultural ences. University of Wisconsin, Madison; search Grant R00801060 from Environmental tion Agency, Washington. D.C.

accepted

Madison.

degrades mexacarbate in the rat intestine by its stability against heat and protease and an acidic pH for a maximum activity.

and spectroscopic identification identified to be flavoprotein.

systems metabolites.

Wisconsin.

Life Sciand by reProtec-

67 0048-3575/79/010067-12$02.00/O Copyright a 1979 by Academic Press, Inc. All rights of reproduction in any form reserved

ESAAC

68

AND

Here we report the results of experiments leading to partial purification and identification of this reductive system from both rat intestine and liver tissues. Optimum incubation conditions and cofactor requirements were also investigated. MATERIAL

AND

METHODS

Tissue preparation. The rat liver and alimentary canal tissues were utilized as the source of anaerobic degradation system. Male albino rats (Sprague-Dawly, 200 g) were purchased from Rolfsmeyer Farm, Madison, Wisconsin. The front portion of the alimentary canal (i.e., small intestine) was excised, cut longitudinally, and the contents were removed. Intestine and liver tissues were washed thoroughly in phosphate buffer NaH2P04/Na2HP04, pH 6.0 (0.02 M) three times. Homogenization was carried out in a Teflon-glass homogenizer at the tissue to buffer ratio of 1 to 2 (wet wtlvol). If not specified otherwise, tissue homogenates were centrifuged for 10 min at 8OOOg followed by a 1-hr centrifugation at 20,OOOg. The resulted supernatant (i.e., 20,OOOg supernatant) was diluted with phosphate buffer to yield a concentration of 36 mg fresh tissue equivalent per milliliter. Incubation mixtures consist of 1 ml of the diluted 20,OOOg supernatant, with the cofactors and/or inhibitors added in buffer, and the volume was adjusted to 1.2 ml with buffer. Incubation was carried out in Thunberg tubes under anaerobic conditions. This was accomplished by successive evacuation and nitrogen flushing three times. The tubes were then incubated in a metabolic shaker for 2 hr at 37°C under laboratory light (approximately 80 fc for 20 min during sample preparation and 12 fc during incubation). In some cases, tubes were incubated in the dark to study the effect of light in the presence and the absence of oxygen. Protein content was estimated according to the Lowry method (5) using bovine serum albumin as the standard. Gel-filtration chromatography of the

MATSUMURA

20,OOOg supernatant. Preliminary studies showed that the degradation activity of the 20,OOOg supernatant was not appreciably altered, when it was subjected to hydrolysis by proteolytic enzymes such as crychymotrypsin or protease. Hence, to purify protein(s) responsible for the degradation activity two approaches were made; namely, protease hydrolysis followed by gel filtration on a Sephadex column. The 20,OOOg supernatant was subjected ta protein hydrolysis by using 2 mg of protease (Sigma Chemical Co.) per milliliter supernatant for 1 hr at 37°C. The resulting hydrolysate was then centrifuged for 20 min at 20,OOOg. A Sephadex column (K 15/!90) was filled by the gel slurry (G-75) in phosphate buffer. The sample was introduced to the top of the gel bed and was eluted with phosphate buffer (pH 6.0). The eluate was collected in 5-ml fractions and was monitored by ultraviolet absorption at 260 or 280 nm, and the degradation activity was measured individually for each fraction using water-soluble 14C-degradation products of mexcarbate as the parameter. Reference proteins with different molecular weights were used to estimate the approximate molecular weight of the active protein(s) separated by gel filteration (6). Cofactors and inhibitors. The partially purified active proteins were subjected to in vitro studies using different cofactors and inhibitors. The incubation mixture consisted of 2.5 ml of the eluate plus cofactor and/or inhibitor added in buffer solution (0.2 ml). Two cofactor groups were assayed for possible stimulation of mexacarbate degradation under different incubation conditions. The first group was the nicotinamide adenine dinucleotide cofactors both in reduced and oxidized forms, namely, NAD, NADP, NADH, and NADPH. Also, an NADPH-generating system was examined. The system consisted of glucose 6-phosphate 6 mg, NADP 3 mg, and glucose-6-phosphate dehydrogenase 16 units per tube. The second

ROLES OF FLAVOPROTEINS

IN DEGRADATION

cofactor group studied was the flavin cofactors: flavin mononucleotide (FMN).’ flavin adenine dinucleotide (FAD), and riboflavin (RbF). All of the above mentioned biochemicals were obtained from Sigma Chemical Company, St. Louis, Missouri. Inhibitors and ions tested were Nethylmaleimide, mersalyl acid, iodoacetate, ethylenediamine tetracetate disodium salt (EDTA), mercuric chloride, potassium cyanide, protoporphyrin IX dimethyl ester (PP), DFP, and p-chloromercuribenzoate (PCMB). Substrate and radioassay. [14C]Mexacarbate (Zectran, 4-dimethylamino-3,5l’4Clxylyl N-methylcarbamate) and its authentic metabolites were provided by Dow Chemical, Inc. [14ClMexacarbate was further purified on thin-layer chromatography (tic) using benzene-methanol (95:5) and silica gel HFzti. It has a specific activity of 8.62 mCi/mmol. Stock solution (lop3 M) of the radioactive compound was prepared in ethyl alcohol and 10 ~1 of this solution (approximately 100,000 cpm) were used per each incubation system. Following incubation, the contents were quantitatively transferred to 15ml culture tubes by using diethyl ether as a rinsing agent and extracted with ether. In routine assays the increase in the radioactivity in the aqueous phase after ether extraction was adopted as a criterion for degradation activities. Water aliquots (0.5 ml) were counted by using a Packard liquid scintillation counter with 10 ml of the counting solution. The ether layer was subjected to tic analysis, using oneand two-dimensional chromatography and four different solvent systems; benzenemethanol (95:5), chloroform-methanol (99: I), ether-hexane (4: I), and chloroform-acetonitrile (4: 1). An autoradiography technique with Kodak No-screen X-ray ‘Abbreviations used: FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; RbF; riboflavin; PP, protoporphyrin IX dimethylester; DFP. O,O-diisopropyl phosphorofluoridate; PCMB. p-chloromercuribenzoate; tic. thin-layer chromatography.

OF MEXACARBATE

IN RATS

69

film along with cochromatographed authentic reference compound on tic was employed in identifying the major degradation product. The major product formed was isolated by using the above tic systems and characterized by NMR analysis in CsD, (Brucker spectrospin, 90 MHz). For each set of experiments, two different incubation mixtures were used as references, control and blank. Control samples refer to incubation mixtures containing the substrate and the tissue preparation without any cofactors. The blank samples contain the tissue preparation to which the substrate is added after incubation and immediately extracted with ether (i.e., a 0-min incubation). The data, illustrated in the following tables and figures, represent the actual degradation activities, since the blank values were subtracted. These data are averages of two to four independent experiments; each carried out on tic chromatogram to the extent of 6-9%. Unless stated otherwise, degradation activity on mexacabate is expressed in percentages of originally added [14Clmexacarbate. RESULTS

Degradation Activity in 20,OOOg Supernatant from Intestine and Its Characteristics

Using rat intestine 20,OOOg supernatant under an anaerobic condition, [‘“Cl mexacarbate was found to be degraded to relatively polar compounds as judged by the increase in radioactivities in the aqueous phase. The water-soluble radioactive products increased significantly by the addition of FAD. The amount of radioactivity that remained in the aqueous phase ranged from 7 to 12% of the original dose of mexacarbate. The following treatment did not alter the degradation activity of the intestinal 20,OOOg supernatant toward [‘*Clmexacarbate: heating the supernatant to 90°C for 10 min prior to the addition of the substrate in the presence of FAD, and addition of streptomycin at 1 ppm concen-

70

ESAAC AND MATSUMURA

tration to eliminate bacterial contaminants (Fig. 1). Incubation of the 20,OOOg supernatant with either a-chymotrypsin or protease had no effect on the degradation activity. Protoporphyrin, alone or in the presence of Fe3+ ions, failed to stimulate the anaerobic degradation of [14Clmexacarbate. Addition of PCMB, DFP, CN-, or Hg2+ (10d4 M) caused a 20 to 90% inhibition of the degradation activities with respect to the amount of radioactivity produced in the aqueous phase (Fig. 1); whereas other -SH inhibitors such as N-ethyl maliemide and mersalyl acid caused a stimulation of degradation activity. Replacement of the cofactor FAD by NADPH or the NADPHgenerating system caused a significant decrease in mexacarbate degradation. The simultaneous addition of both FAD and NADPH to the incubation mixture caused an inhibition of mexacarbate degradation (Fig. 1). These preliminary studies clearly indicate that the rat intestinal tissues exhibit degradation activity toward mexacarbate which is stimulated by FAD. Mexacarbate degradation by the intestinal 20,OOOg supernatant was then investiDegradation Activity,

%

gated under various incubation conditions, using the amount of radioactivity present in the aqueous phase as a criterion for the rate of degradation activity. To study the effect of pH, phosphate buffer was used to adjust the pH range from 5.0 to 8.0, while for pH 4.0 the acetate buffer was utilized. As the pH of the incubation mixture increased from 4.0 to 8.0, there was a gradual but consistent increase in the water-soluble products, though the increase was observed both in the presence and absence of FAD. However, when the difference between FAD treated and nontreated tests was calculated, it became apparent that stimulatory action of FAD had the optimum at pH 6.0 (Table 1). With respect to the amount of FAD in each incubation mixture, it was found that the minimum amount of FAD required to reach the highest level of stimulation of the degradation reactions was 10 pg per assay. Increasing the amount of protein per each incubation mixture also caused an increase in the mexacarbate degradation to polar compounds (Fig. 2). This increase was roughly proportional to the amount of protein. The rate of mexacarbate degradation was stimulated significantly by the increase in reaction temperature. This rate of increase was small at low temperatures, 10 to 3O”C, but was greater at higher temperatures, up to 60°C (Fig. 3). This

FAD I

Protoporphyrln

MProtoporphyrin

TABLE

+ Fe3’

!

Protoporphyrln Chymotrypsln

+ FAD

Effect Intestine

Proteasel !

by the of FAD

Heat

Control

Control + FAD

PH

[Al

PI

Ratio A/B

4.0 5.0 5.6 6.0 6.6 7.0 7.6 8.0

1.1 0.8 0.7 0.5 0.8 1.7 4.4 7.9

1.0 4.0 4.0 6.1 7.2 7.6 11.5 13.6

0.9 5.0 5.7 12.2 9.0 4.5 2.6 1.7

Dialyzed Cyanide -

D

Degradation in the Presence

‘T-Water-soluble degradation products, percentage as of initial

Streptomycin

I

-

of pH on Mexacarbate 20,OOOg Supernatant

1

DFP PCMB Hg++

FIG. 1. Degradation

activities on [‘*Clmexacarbate by the rat intestine 20,OOOg supernatant under different treatments. Activity is expressed in percentages of initially added mexacarbate appearing as ‘Y’water-soluble polar products.

ROLES

OF

FLAVOPROTEINS

IN DEGRADATION

OF MEXACARBATE

71

IN RATS

8 p

1

i

I

30 i

/

1

40 80 120 160 Concentration. mgTissue FIG. 2. Relation between the amount of protein added and mexacarbate degradation by the intestine 2O.OOOg supernatant. For activity see Fig. I.

trend was observed only in the presence of FAD but not in its absence. In the absence of the flavin cofactor, the amount of radioactivity in aqueous phase never exceeded 2.2% even at 60°C. As was the case with the increase in temperature, the increase in substrate concentration likewise resulted in the increase in the apparent rate of mexacarbate degrada-

50 Concentration, FIG. 4. Rate of anaerobic as influenced by substrate see Fir. I.

100 nanomoles

mexacarbate concentration.

degradation For ac’tivit?

tion (Fig. 4). For a range of 200-fold substrate concentration, the amount of polar products gradually increased. However, when these data were treated by the Lineweaver-Burk method a curve rather than a straight line was obtained (Fig. 5). the fact

> \

Temperature FIG. 3. Effect of temperature on mexacarbate degradation to water soluble products by the intestine 20,ooOg supernatanr. For activity see Fig. 1.

FIG. 5. Linewearser-Burk plot showing substrate effect on FAD-stimulated degradation activity of the 20,OOOg supematant on mexacarbatr.

72

ESAAC

AND

MATSUMURA

Qvoprotein

2.0

a 0 E -

c I.( IO

20 Fractions

30

5:

.n

40

FIG. 6. Column gel-filtration chromatography using Sephadex G-75 for the rat intestine 20,OOOg supernatant before and after protease treatment. (0) 20,OOOg

supernatant;

(A)

proteolytic

hydrolysate. I

suggesting that the anticipated reaction is not simple.

degradative

Purification of the Active Protein from the Liver and Intestine Elution patterns on column gel filtration of the 20,OOOg supernatant of rat intestine (Fig. 6) or liver (Fig. 7) show that there were two major protein peaks which align with the degradation activities on mexacarbate for each tissue preparation. Treatment of the 20,OOOg supernatant with protease enhanced the degradation activity particularly at peak II (Fig. 6). Since peak II is relatively uncontaminated by nonactive

4 FAD

a

I

I

I

300 200 Wavelength, nm FIG. 8. Ultraviolet tion

(32

pglml)

spectra and

FAD

offlavoprotein (IO pg)

prepara-

in phosphate

buffer.

proteins, the 20,OOOg supernatant was first incubated with protease and subjected to gel filtration with Sephadex G-75 to yield a partially purified fraction, peak II. As a result of spectroscopic analyses it was found that peak iI contained a large titer of flavoproteins as judged by its uv spectra (Fig. 8) and fluorescence spectra (Fig. 9). The frac.t,”

Excitation

80-

5 C s 60s :: p! 402 .$ G E

20-

300 Fractions FIG. 7. Column

gel-filtration

Sephadex G-75 for the rat before and affer protease wpernatant; (A) proteolytic

chromatography liver 20,OOOg treatment. hydrolysate.

500

600

Wavelength, nm

using supernatant (0) 20,OOOg

400

FIG.

9. Excitation

spectra of theffavoprotein tine in 100 mM acetic

and

fluorescence preparation

acid.

emission from

rat intes-

ROLES OF FLAVOPROTEINS

IN DEGRADATION

tion is, hereafter, referred to as the flavoprotein preparation. From the position of the peaks resolved in Figs. 6 and 7, it is evident that peak II is entirely a product of protease hydrolysis. The molecular weight of the protein(s) associated with peak II was estimated to be around 6000 to 10,000. In case of the liver preparation (Fig. 7) the two peaks were relatively broader than those from the intestinal preparation, indicating that the liver preparation contained higher titer or more types of proteins than the corresponding intestine preparation.

TABLE Mexacarbate

Degradation under

Incubation condition and cofactor added Anaerobic in light None FAD FMN RbF Aerobic in light None FAD FMN RbF Anaerobic in dark None FAD RbF Aerobic in dark None FAD RbF

by the Different

Mexacarbate remaining (%)

73

IN RATS

Addition of a flavin cofactor significantly stimulated the degradation reactions. With respect to the flavin cofactors, both riboflavin and FMN were equally active in stimulating mexacarbate degradation and were significantly more effective than FAD. When the incubation was carried out under laboratory light, the extent of degradation was virtually unaffected by the presence or absence of oxygen. On the other hand, if the incubation is carried out in the dark, the presence of oxygen caused an almost complete inhibition of mexacarbate degradation. These data indicate that degradation of mexacarbate by the flavoprotein preparation is an anaerobic-type reaction, and that under light and in the presence of oxygen unrelated, probably photochemical reactions are taking place. In the case of the liver flavoprotein preparation, similar results were obtained with respect to the influence of various in-

Anaerobic Degradation Activity by the Flavoprotein Preparation Mexacarbate degradation by the intestinal flavoprotein preparation was carried out under various incubation conditions (Table 2). Mexacarbate was least affected when it was incubated without any added cofactor.

[‘VI

OF MEXACARBATE

2

Rat

Intestine

Incubation

Flavoprotein

Preparation

Conditions

Degradation products (%l Watersoluble

Desmethyl”

--.Other ether soluble*

82 55 15 11

1 9 25 26

6 17 18 23

4 12 35 33

81 72 7

2 2 27

4 7 14

5 11 44

6

31

19

36

1 5

I 2

92 86 8 94

91 88

1 2 30

I 1 2

37

1 2 4

20

1 3 3

” Desmethyl refers to the major metabolite desmethylmexacarbate. h Other ether soluble products refer to degradation products (four to six) separated by tic analyses.

74

ESAAC AND MATSLJMURA

cubation conditions (Table 3). The difference was, however, that the extent of degradation was greater in the liver than in the intestinal preparations. Each flavin cofactor added resulted in the formation of water-soluble products at significantly greater levels in preparations from the liver than that from the intestine. Regardless of the flavin cofactor used or the protein source, 4-N-desmethylmexacarbate was the major ether-soluble product (Tables 2 and 3). In each of the intestine and liver flavoprotein preparations, heating of the enzyme solution prior to the addition of substrate and flavin cofactor did not alter the extent of mexacarbate degradation to water and ether-soluble products. The degree of anaerobic degradation of mexacarbate by various flavin cofactors was similar to the above mentioned results for intestine and liver preparations. The addition of NADPH caused little stimulation when present alone; whereas it TABLE [‘4C]Mexacarbate

caused an appreciable inhibition when added along with riboflavin (Table 4). Similarly, the presence of both riboflavin and the NADPH-generating system did not stimulate the anaerobic reactions toward mexacarbate as did the addition of riboflavin only. In fact, none of the nicotinamide adenine dinucleotide cofactors, oxidized or reduced, showed any appreciable degree of stimulation of anaerobic degradation of mexacarbate (Table 4). The same trend was observed when the NADPH-generating system was added alone to the incubation mixture. For example, greater than 80% of the initial mexacarbate was recovered as unchanged, when the reduced cofactors (NADH, NADPH, and NADPH-generating system) were used. On the other hand, the addition of the oxidized cofactors (NAD or NADP) caused a relatively higher rate of mexacarbate degradation than the corresponding reduced cofactors. The anaerobic degradation of mexacarbate was almost identical when the 3

Degradation by the Rat Liver Flavoprotein under Different Incubation Conditions

Preparation

Degradation products (%)

Mexacarbate remaining (%)

Watersoluble

Anaerobic in light None FAD FMN RbF

78 42 5 5

2 17 56 51

4 23 21 18

9 11 11 19

Aerobic in light None FAD FMN RbF

78 58 4 4

2 11 45 48

4 8 12 15

3 10 26 20

Anaerobic in dark None FAD RbF

88 78 9

3 3 21

3 12 33

1 2 32

Aerobic in dark None FAD RbF

92 84 88

2 8 4

1 3 3

Incubation condition and cofactor added

n See Table 2.

Desmethyl”

Other ether solublea

ROLES OF FLAVOPROTEINS

IN DEGRADATION TABLE

Effect

of the

Nicotinumide

Adenine Dinucleotide by the Rat Liver

None Riboflavin (RbF) RbF + NADPH RbF + NADPHgenerating system NAD NADP NADH NADPH NADPH-generating system

75

IN RATS

4

Cofactors Navoprotein

on the Anaerobic Preparation

Degradation

of Mexaccrrhatr -

Degradation products (%,)

Mexacarbate remaining (%%)

Cofactor added

OF MEXACARBATE

Watersoluble

Other ether soluble”

Desmethyl”

78 5 63

2 51 8

4 18 11

9 19 11

64 75 72 80 84 84

2 1 2 1 1 1

I1 10 8 4 4 5

16 7 II 8 4 3

” See Table 2.

incubation is carried out under carbon monoxide instead of nitrogen (Tables 3 and 5). Mexacarbate degradation was inhibited to varying degrees by the addition of EDTA, cyanide, or iodoacetate. A significant decrease in the amount of watersoluble products was experienced in the presence of these chemicals (Table 5). Intracellular Distribution of the Anaerobic Degradation Activity on Mexacarbate in Intestine and Liver Tissues Incubation was carried out for the following tissue preparations: 800g supernaTABLE Effect

of Ceriain

Inhibitors

tant, 20,OOOg supernatant, proteolytic hydrolysate, and flavoprotein preparation (Table 6). For each of the intestine and liver tissues, anaerobic degradation activity was more clearly recognized in the flavoprotein fraction than in any of the other preparations assayed. The extent of anaerobic degradation of mexacarbate by liver preparations is greater than the corresponding intestine preparations. On the other hand, the specific activity, in terms of nanomoles of mexacarbate degraded per milligram protein in 2 hr, was severalfold greater for the intestine preparations than those of the 5

and Carbon Monoxide on the Anaerobic Degradation in Light. by Rat Liver Flavoprotein Preparation

of Mexacarbate,

Degradation products (%) Cofactor and/or inhibitor Control RbF EDTA only EDTA + RbF Cyanide only Cyanide + RbF Iodoacetate only Iodoacetate + RbF CO only CO + RbF (’ See Table 2.

Mexacarbate remaining (%i) 78 5 76 49 68 12 58 19 50 2

Watersoluble 2

51 3 12 2 20 5 26 3 58

Ether-soluble” Desmethyl 4 18 9 24 11 13 3 2 24 11

_-Others 9 19 5

12 12 48 27 46 16 22

ESAAC AND MATSUMURA

76

TABLE Specific

Activity

of the Anaerobic Degradation and Liver Tissue

6 of Mexacarbate Preparations

by Diflerent

Intestine

Specific activity (nm mexacarbate degraded/mg protein/2 hr) Tissue preparation and source

Preparation only

Preparation + FAD

Preparation + riboflavin

8OOg supematant Intestine Liver

2.8 4.1

6.9 7.8

7.8 11.4

20,OOOg supernatant Intestine Liver

2.1 1.4

4.8 1.5

7.2 6.6

3.3 5.1

6.4 5.6

14.7 8.0

10.9 4.3

27.2 11.5

53.7 19.1

Proteolytic hydrolysate Intestine Liver Flavoprotein preparation Intestine Liver

liver (Table 6). As mentioned earlier, the addition of a flavin cofactor, regardless of the tissue preparation tested, enhanced the anaerobic degradation of mexacarbate. Degradation of ~14ClDesmethylmexacarbate by the Flavoprotein Preparation Throughout the experiments the increase in water-soluble metabolites has been used TABLE Anaerobic

Incubation conditions Control Light Dark

Degradation

of [‘Y’]De~~ethylmexacarbate

Desmethyl remaining (%‘o)

as the criterion for degradation, while desmethylmexacarbate is consistently the major ether-soluble degradation product detected. Hence, experiments were carried out using this major product as the substrate in place of mexacarbate to provide the evidence that these two phenomena are related (Table 7). Anaerobically, degradation of desmethylmexacarbate by the rat intestine flavoprotein preparation is almost 7 by the Rat Intestine

Flavoprotein

Preparation

Degradation products (%) Water-soluble

Ether-soluble

10 12

26 25

56

15 14

29 30

Riboflavin Light Dark

16 18

59 58

25 24

Blank Light Dark

71 73

12 12

17 15

FAD Light Dark

64

63 58

ROLES

OF FLAVOPROTEINS

IN DEGRADATION

identical in the presence or absence of light indicating that the reaction is not a photochemical one. As was the case with degradation of mexacarbate, desmethylmexacarbate degradation is also flavin dependent. Riboflavin addition caused a significant increase in the amount of watersoluble products. The degradation of either mexacarbate or desmethylmexacarbate, in the presence of FAD or riboflavin, showed the same trend with regard to the amount of polar products detected (Tables 2,3, and 7). Chemical Identification of the Major Ether-Soluble Product In order to prepare enough quantity of the major ether-soluble product, 180 ml of the flavoprotein preparation from rat intestine were incubated with 6 mg of cold- and 60 pg of labeled-mexacarbate in the presence of FAD. Incubation was carried out for 4 hr in a 125ml Enlenmeyer flask (60 ml) to which a manifold was attached to permit evacuation and nitrogen flushing. The ether extract was analyzed by tic using

hkxacarbate

Major Product

II

-lL---_ii, I

8

I

I

6

I

I

4

1

1

2

1

PPm

Proton magnetic resonance spectrum mexacarhate, 4-N-desmethylmexacarbate and major ether-soluble product: taken in CsD6. FIG.

10.

of the

OF MEXACARBATE

IN RATS

77

OS-mm thick plates. The major band was purified by three successive tic analyses using different solvent systems. The Ndesmethyl residues (2-3 mg) were transferred to an NMR tube using 0.3 ml of C6Ds and 10 ~1 of TMS as reference. The nuclear magnetic resonance spectrum was identical to the reference compound, 4-N-desmethylmexacarbate (Fig. 10). DISCUSSION

It is clear from the result of our experiment that the flavin-flavoprotein system is different from the NADPH-requiring reductive system described by other workers (24). The former system is heat stable, insensitive to CO. and does not require NADPH for its activity. On the other hand, it is not possible to deduct from our data how much of reductive activities found by other workers in the presence of flavin cofactor(s) are from the flavin-flavoprotein reactions. As for the mechanisms of degradative activities of flavin-flavoprotein system we must first consider the fact that mexacarbate degradation occurred under all possible combinations of light or dark and presence or absence of OZ. In the presence of light, mexacarbate degradation is practically identical, whether the incubation is carried out aerobically or anaerobically. It is highly probable that in the light photoreduction of flavins takes place under both aerobic and anaerobic conditions. It has been known that in photosensitized reactions, triplet flavin transfers electronic energy to molecular oxygen to give singlet oxygen, a powerful oxidant. This route has been implicated in flavin-sensitized photooxidation of purines, aromatic amines. and phenols (8). Under anaerobic conditions, flavins are reduced in the presence of light by a number of electron donors including amines (9). amino acids (IO), and NADH (11). On the other hand, mexacarbate degradation took place, in dark, only in the absence of oxygen. Hence, it follows that its degradation should be regarded as a reductive reaction. At present the most logical

78

ESAAC

AND

explanation for this reductive reaction is that flavoproteins act as the proton donors to reduce flavin cofactors which in turn reduce mexacarbate, mainly, to 4-N-desmethylmexacarbate. There are two observations to support the above assumption: first, acidic conditions increase the availability of protons to favor the reaction, and second, the minimum concentration of FAD to give a maximal degradation was in the same order of molarity as that of the substrate, mexacarbate. The involvement of flavoproteins is also supported by the fact that they are known to withstand the heat and are released by protease treatments. In that case, these flavoprotein involving degradation systems must be considered as essentially nonenzymatic. As for its significance in degradation of pesticidal chemicals in vivo, we have already shown (11) that DDT is converted to TDE by the same intestinal flavin-flavoprotein system. Since the above process is the essential first step for the degradation of DDT eventually to DDA, we have concluded that the system could certainly play a significant role in pesticide degradation in animals. The fact that the system is present in the intestine, where anaerobic condition does exist in the absence of light points to the possibility that such system could operate in vivo for DDT degradation. On the other hand, the major pathway for mexacarbate metabolism in viva is through hydrolysis followed by conjugation of the resulted phenolic compounds, since the excreted metabolites were found to be either free phenols or their conjugated derivatives (12, 13). The fact that both rat microsomes under oxidative conditions (14) and the flavoprotein system reported here under reductive conditions are capable of producing the desmethyl derivative as a major product in vitro raises the question regarding the relative importance, if any, of each system in vivo. While it is reasonable to assume that the comparatively anaerobic conditions of the gastrointestinal tract are ideal for reduction of foreign chemicals in-

MATSUMURA

gested, the impact of this flavoproteinflavin cofactor system on the detoxification of mexacarbate in vivo is yet to be accurately assessed. REFERENCES I. J. R. Gillette, in “Handbook of Experimental Pharmacology” (B. B. Brodie, and J. R. Gillette, Eds.), p. 349, Springer-Verlag, Berlin/ New York, 1971. 2. K. A. Hassall, Reductive dechlorination of DDT: The effect of some physical and chemical agents on DDD production by pigeon liver preparation, Pestic. Biochem. Physiol. 1, 259 (1971). 3. J. R. DeBaun and J. J. Met-m. Sulfoxide reduction in relation to organophosphorus insecticide detoxification, Science 191, 187 (1976). 4. S. Khalifa. R. L. Holmstead, and J. E. Casida, Toxaphene degradation by iron (II) protoporphyrin system, J. Agr. Food Chem. 24,277

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