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The conversion of pyrrolizidine alkaloids to N-oxides and to dihydropyrrolizine derivatives by rat-liver microsomes in vitro
Chem-Biol Interacttons Elsewer Publishing Company, Amsterdam Printed an The Netherlands
383
THE C O N V E R S I O N OF P Y R R O L I Z I D I N E A L K A L O I D S TO N-OXIDES A N D TO D I H Y D R O P Y R R O L I Z I N E D E R I V A T I V E S BY R A T - L I V E R M I C R O S O M E S IN VITRO
A R MATTOCKS AND I N H WHITE
M R C Toxtcology Umt, Medtcal Research Counctl Laboratories, Woodmansterne Road, Carshalton, Surrey (Great Brltatn) (Received January 5th, 1971) (Rewslon accepted February 4th, 1971)
SUMMARY
The enzymatic conversion of pyrrohzldlne alkaloids to dlhydropyrrolazlne derivatives ('pyrrole derivatives') and to N-oxides has been investigated. In rat liver both reactions were catalysed by an enzyme present in the mlcrosomal fraction, typical of the mixed-function oxldases Thus the conversion of alkaloids to pyrrole derivatives and to N-oxides required oxygen and reduced N A D P and was activated by Mg 2+, Mn 2+ or Ca 2+ at concentrations of 5 10 -3 M The liver mlcrosomal system was less active an the female rat than an the male and was inhibited by carbon monoxide and by SKF 525A The conversion of retrorslne to both pyrrohc metabohtes and to N-oxides was significantly reduced by pretreatment with phenobarbltone or DDT, but the Induction of pyrrole derlvatwes was greater than that of N-oxide The conversion of retrorslne to pyrrole derivatives was lower an liver mlcrosomal fractions from rats which were starved or fed on protein-free d~ets Differences between the rates of formation of pyrrole derivatives and N-oxides followed the ancluslon of nlcotmamlde m the incubation mixtures containing macrosomes from untreated rats, or substitution of a carbon monoxide-oxygen mixture for air during the incubation period The Km values towards the formation of pyrrolic denvatwes and N-oxides from retrorslne from normal rat-laver mlcrosomes were similar No conclusive evidence was found to establish whether N-oxides were intermediate m the formation of pyrrole derivatives. However, pyrrole denvatwes could not be detected when N-oxides were added to the hepatic mlcrosomal system m vztro Rat-hver mlcrosomal enzymes converted a number of alkaloads havang either 1,2-dehydropyrrohzadlne or saturated pyrrohzldlne ring structures to pyrrole denvatwes The pyrrole derlvatwes from these two groups of alkaloids had different chemical properties Only alkaloads in the first group were hepatotoxlc, and in general the more toxic alkaloids in th~s group were Abbreviation NTP, nontoxic plasticizer
Chem-Blol Interacttons, 3 (1971) 383-396
384
A R. MATTOCKS, I N H WHITE
those from which the greatest amounts of pyrrole derivatives were produced m vitro, although there were exceptions The enzymatic conversion of retrorsme or monocrotahne to pyrrohc derivatives could not be demonstrated in lung mlcrosomal fractions
INTRODUCTION
The action of many pyrrohzldme alkaloids in bringing about both acute and long-term changes in the livers of experimental animals has been described 1'2. These alkaloids are metabohsed by a variety of pathways such as hydrolysis, hydroxylatlon and N-oxidation (ref. 3, p. 215). Alkaloids having a 1,2-dehydropyrrohzldlne ring structure undergo conversion in the liver to pyrrole-hke derivatives *'5 and it has been suggested that such reactive derivatives may be responsible for toxic effects4 . The hepatic microsomal system has been imphcated in this reaction 6 and the conversion of hehotrldlne-based alkaloids to dlhydropyrrohzine derivatives m vitro has recently been demonstrated using rat hepatic mlcrosomal preparations 7. The work to be described examines some of the propemes of the enzymes in rat hver which are responsible for converting the alkaloids to N-oxides and pyrrole derivatives The efficiency of the system m the productmn of pyrrole derwatwes from a number of dlfferent pyrrohzidme alkaloids m vitro is compared with the published acute tOXlCmes of the alkaloids tn vtvo The effects of pretreatmg rats in a number of ways, on the mlcrosomal metabolism of these alkaloids m vitro are also described. MATERIALS AND METHODS
Chemicals were obtained from B D H L t d , unless otherwise mentmned N A D , NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Boehrlnger Corporation (London) Ltd Preparation of hver fractions
Albino Porton rats (150-200 g, males except where otherwise stated) were killed by a blow on the head and exsangumated, normally between 10 00 and 11 00 h. The hvers were quickly removed and placed in ice-cold 1 15 ~ KC1. All the remaining procedures were carried out at 0-4 ° unless otherwise indicated. After being blotted, the livers were weighed, minced with scissors and 33 ~ (w/v) liver homogenates prepared in 1.I 5 ~ KC1 using a pestle-type homogemser with 0 02-inch clearance 8. The homogenate was centrifuged at 12 000 g for 20 m m in a MSE HighSpeed 18 centrifuge. The precipitate was resuspended m 1 15 ~ KC1 equal in volume to that of the original homogenate, while the supernatant was further centrifuged at 105 000 g for 60 mln in a Splnco Model L ultracentrifuge to give a mlcrosomal pellet and a supernatant (soluble) fraction. When washed mlcrosomes were required, the pellet was resuspended in the original volume of 1.15 ~ KC1 and resedlmented as before The mlcrosomal pellet was finally resuspended in a volume of 1 15 ~ KC1 Chem-Btol Interactions, 3 (1971) 383-396
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVES AND N-OXIDES
385
such that 1 ml was equivalent to 1 g of hver wet weight. Mlcrosomal protein was estimated by the method of LOWRY et al 9 with bovine serum albumin as the protein standard
Preparation of lung microsomal fractton Rats were decapitated and thoroughly exsangulnated, and the lungs from 6-8 animals were removed as quickly as possible and pooled in ice-cold 1 15 ~ KC1. After being homogenised in the same way as the hver, the suspensmn was filtered through a single layer of surgical gauze. Subsequent procedures for cell fractIonatlon were carried out in a manner identical to those already described for rat liver
Assay procedures Incubation mixtures of 3 ml final volume consisted of 0 25 M phosphate buffer (pH 7 4), 14 m M MgC12, 0 25 m M N A D , 0 22 m M N A D P , 4.0 m M glucose-6-phosphate and 0.5 I.U./ml glucose-6-phosphate dehydrogenase Substrates were pure crystalhne pyrrohzldine alkaloids neutrahsed wlth an equivalent of HC1 or pyrrohzldme N-oxides at concentrations of 3 3 mM, unless otherwise mdxcated. Following equlhbratton at 37 °, reactions were started by the a d d m o n of 0 5 ml of the mtcrosomal suspensxon (containing about 8 mg mlcrosomal protein) Flasks were shaken at 100 strokes per mln in air for 10 m m unless otherwise indicated Reactions were stopped with ethanol N-Oxide metabohtes were chemically converted to pyrrole derivatives which were then estimated using Ehrhch reagent. Pyrrohc metabohtes were estimated directly by an Ehrhch reactmn Detads of these procedures have been described ~°.
Determmatton of Km values Apparent K,, constants were determined by means of Woolf plots (see ref 11) ([S]/v vs [S]), best-fitting lines being drawn by the method of least squares A range of alkaloid concentrations from 0 1 m M to 1 m M was employed.
Effect of mhtbttors The action of a number of lnhlbltors was investigated by adding them to the reaction mixture immediately prior to incubation. Compounds tested included fldlethylamlnoethyl dlphenylpropylacetate (SKF 525A; Smith, Klyne and French L t d ) (5. 10 -4 M), K C N (5 10 -3 M), and nlcotmamide ( 1 . 4 . 1 0 -2 M) In experiments where air was not employed as the gas phase, the appropriate gas mixture was passed through flasks containing the reaction mixture and mlcrosomes but no alkaloid, at 4 ° After 5 mln, the flasks were equlhbrated at 37 ° and the reaction started by the addmon of alkaloid A gas flow of 500 ml/mln through the flasks was maintained for the duration of the experiment.
The effect of cations The actions of the chlorides of Mg z+, Mn 2+, Ca z÷, Co 2÷ and Cu 2÷ were tested by their Inclusion m the reaction mixtures at concentrations of 5 10 -4 M and 5 • 10 -3 M Procedures were the same as described above Washed mlcrosomes were used. Incubatmn mixtures contained 0 25 M Hepes (N-2-hydroxyethylpxperChem-Btol Interactions, 3 (1971) 383-396
386
A. R. MATTOCKS, I N. H. WHITE
azme-N-2-ethanesulphonlc acxd) (Sigma Chemxcal Co ) buffer 12, 3 3 m M retrorslne, 1 m M N A D P H (chemically reduced), 0.25 m M N A D and 1 15 % KCI to give a final reactmn volume of 3 ml
Diets and predosmg Rats were normally fed ad hb. a M R C 41B diet 13 with free access to water at all times. Sodium phenobarbitone (100 mg/kg) was administered either by lntraperitoneal rejection in saline, 72, 48 and 24 h before the rats were killed, or as a 0.1% solution in the drinking water for 7 days 14. The followmg compounds were administered by lntraperltoneal rejection at the indicated times before the rats were killed: 3-methylcholanthrene (Koch-Light Laboratories), 20 mg/kg in trioctanoin (Eastman) at 72, 48 and 24 h; 1,1,1-mchloro-2,2-bls(p-chlorophenyl)ethane (DDT) (Geigy), 75 mg/kg in trioctanoxn at 72 h; amlnoacetonltrde blsulphate (Koch-Light Laboratories), 100 mg/kg m sahne 48, 24 and 1 h; and retrorsme hydrochlonde, 35 mg/kg in saline at 20 h NTP*, 2000 mg/kg, was administered orally as a 50 % solutmn in arachls oil, 60 h before the rats were killed. Rats were also maintained on diets of" Analar granular sucrose for 2 days; on the protein-free diet described by MCLEAN AND MCLEAN ~5 for 3 days; or starved for 2 days before being killed. RESULTS
Table I shows a comparison between enzymatic activities associated with the conversion of retrorslne to pyrrole derivatives and to N-oxides in subcellular fractions of rat liver The recoveries, expressed as percentages of the activities found in the original liver homogenate, appeared to be similar for both reactions. The highest specific activity for the formation of both pyrrole derivatives and N-oxides was In the mlcrosomal fraction Specific mlcrosomal activities, for the formation of pyrrole derivatives and N-oxides in female rats were respectively about 15 % and 30% those of male rats. The time-courses for the enzymatic conversion of retrorslne to pyrrole denvattves and to N-oxides by a male rat-hver mlcrosomal fraction are shown in Fig. 1. The rates of conversion to both metabolites remained linear for the first 10 mln when both N A D P H and N A D H were present. Omission of N A D H had little effect on the initial enzymatic activities, but both reactions terminated more quickly than when both cofactors were present. No enzymatic conversion of retrorslne to pyrrole derivatives or to N-oxides could be detected in the presence of N A D H alone. A number of cations were examined for their effects on the mlcrosomal conversion of retrorsine to pyrrohc derivatives (Table II). Apart from Cu z÷ which was strongly inhibitory, the remaining cations tested had little effect at concentrations of 5 • 10 . 4 M At a concentration of 5. 10 -a M, however, Mg 2+, Mn 2+ and Ca 2+ all stimulated microsomal activity Experiments designed to investigate the effects of one cation In the presence of another, indicated that in the presence of Mg 2+ either Mn 2+ or Ca 2+ caused further activation, but Mn 2+ and Ca 2+ together did • A mixture o f tn-aryl p h o s p h a t e s (Lot N o N r Chesterfield, U K )
Chem-Blol Interactions, 3 (1971) 383-396
S 1853, C o a h t e Chemical Products L t d , Bolsover,
387
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVES AND N-OXIDES TABLE I RECOVERY
AND
SPECIFIC ENZYMATIC
ACTIVITIES ASSOCIATED WITH
TO PYRROLE DERIVATIVES OR TO N-OXIDES
Fractton
IN VARIOUS SUBCELLULAR
Specific acttvlty (nmoles/mm/mg protein 4- S E ) Pyrrole dertvattve
N- Oxtde
1 06 4- 0 16
0 83 4- 0 04
0 51 1 59 3 20 0 51
0 67 1 14 2 27 0 40
THE
CONVERSION
OF RETRORSINE
FRACTIONS OF RAT LIVER a
Recovery ( % o f mean homogenate acttvtty 4- S E ) Pyrrole N- Oxide derlvattve
Male rats Homogenate Cell debris, nuclei, mltochondrm, etc 12 000 g supernatant Mlcrosomes 105 000 g supernatant
4± 44-
0 0 0 0
10 12 40 15
4444-
0 0 0 0
05 12 19 10
344-2 65 4- 5 33 4- 3 13+1
31 72 34 11
:t:2 b 4- 5b 4- 4 b -{- 1b
Female rats Mlcrosomes
0 54 4- 0 12
0 72 4- 0 07
a Each value represents the mean o f 4 experiments b NO slgmficant difference between the percentage recovery of pyrrole derivative and N-oxide
not activate the system to a slgmficantly greater extent than either 1on by itself. Some compounds known to be mhlbltors of the mlcrosomal drug-metabohslng system were examined for their effects on the metabohsm of retrorsme to pyrrole derivatives and to N-oxides using a rat-hver mlcrosomal preparation (Table III) Incubanons carried out anaerobically, m the presence of SKF 525A, or a carbon monoxide + oxygen mlxture, all resulted m a slgmficant decrease in the amount of pyrrole and N-oxide formed. Substltunon of 100 % oxygen for mr had little effect on the production of pyrrohc metabohtes but significantly stimulated N-oxide formation when measured over o Q.
~ o
~0c
0 ~b
_~ 8C
g E
,o
60
,.0
40
[]
20
o
[]
J
I0
20
.3b 4o 5'0 INCUBATION TIME (MIN)
6~0
Fig 1 Time-course o f the rat hepatic mlcrosomal conversion of retrorsme to pyrrole denvataves and to N-oxides in wtro Pyrrole denvanves produced in the presence o f N A D P H and N A D H (O) m the presence o f N A D P H ([5]). N-Oxade produced m the presence o f N A D P H and N A D H ( O ) , in the presence o f N A D P H (11)
Chem-Btol Interactlons, 3 (1971) 383-396
388
A. R. MATTOCKS, I N. H. WHITE
T A B L E II EFFECT OF VARIOUS CATIONS ON RAT-LIVER MICROSOMAL CONVERSION OF RETRORSINE TO PYRROLIC METABOLITES In vltro a
Enzyme activity ( ~ o f mean control values 4- S E ) at cation concn ( M )
Cation
M g 2+ M n 2+ Ca 2+ Co z+ Cu 2+ MgZ+ + Mn2+ b Mg2+ + Ca2+ b Mg2+ q_ Ca2+ 4- Mn2+ b
5 I0 - 4
5
103 102 104 108 29
115 127 138 70 20 120 127 132
4- 5 4- 1 4- 4 4- 2 4-4
I0 - 3 44444444-
5 15 1 1 1 4 1 4
a Each result represents the m e a n of 2 experiments b MgZ+ c o n c e n t r a t i o n was 1 10 - 2 M
a 10-mm penod. Potassium cyamde proved to have little effect on either system. Nlcotlnamlde was without effect on the conversion of retrorsme to N-oxide but brought about a significant lnhlbttmn of the formation of pyrrohc derivatives Table IV shows the effect of pretreatmg rats with a number of compounds Phenobarbltone and D D T pretreatment both greatly stimulated the hver mlcrosomal conversion of retrorsme to pyrr.ole denvatlves in vitro, while the effect on N-oxide productmn was somewhat less pronounced Pretreatment of rats with ammoacetomtrlle stimulated the productmn of pyrrohc derivatives from retrorsme. NTP, an organophosphorus esterase mhxbltor, also stimulated the formation of pyrrohc metabohtes Retrorslne pretreatment at shghtly less than the acute LDso dose brought about a marked decrease m the hver mlcrosomal pyrrole formatmn The results of experiments using protein-depleted rats showed that lrrespect~ve of the way m which this was achieved, protein depletmn brought about a slgmficant decrease in the hver m~crosomal conversmn of retrorsme to pyrrole derlvatwes. T A B L E III THE EFFECT OF VARIOUS GAS MIXTURES AND INHIB1TORS ON THE In vitro CONVERSION OF RETRORSINE TO PYRROLE DERIVATIVES AND TO N-OXIDES BY RAT-LIVER MICROSOMAL PREPARATIONSa
Compound
Enzyme activity ( % o f mean control values 4- S E ) Pyrrole derivatives
N i t r o g e n (100K) C a r b o n m o n o x i d e -k o xygen (80 20) Oxygen ( 1 0 0 ~ ) S K F 5 2 5 A ( 5 10 - 4 M ) P o t a s s i u m cyanide (5 1 0 - 3 M ) NlCOtlnamlde (1 4 1 0 - 2 M )
20 4- 1 (P < 0 001) c 42 102 35 100 73
4- 3 (P < 0 001) c 4- 4 (N S )c 4-4(P<0001) c 4- 2 (N S )c 4- 2 (P < 0 001) e
Stgmficance b
N-Oxide 22 4- 1 ( P < 0 0 0 1 )
e
68±2(P<0001) e 114 4- 2 (P < 0 01) ¢ 41 4 - 2 ( P < 0 0 0 1 ) e 90 4 - 5 ( N S ) c 98 4- 2 (N S ) c
NS P <001 N S NS NS P < 001
a E a c h result represents the m e a n o f 4 experiments b Pro babilities t h a t differences between rates o f f o r m a t i o n o f p y r r o l e derivatives a n d N-oxides are significant N S , n o t significant c P r o b a b i l i t i e s t h a t differences between e n z y m a t i c activities in the presence of m h l b l t o r s or gas mixtures a n d c o n t r o l s are significant
Chem.-Biol Interactions, 3 (1971) 383-396
389
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVES AND N-OXIDES T A B L E IV
EFFECT OF PRETREATING RATS ON THE LIVER MICROSOMAL CONVERSIONOF RETRORSINE TO PYRROLE DERIVATIVES AND TO N-OXIDES in Htro a Pretreatment
Enzyme acttvtty ( % o f mean control va?ues ± S E ) Pyrrole dertvattves
Phenobarbltone (3 x 100 mg/kg, 1 p ) D D T (1 × 75 mg/kg, 1 p ) 3-Methylcholanthrene (1 × 2 0 mg/kg, l p ) Amanoacetonltrlle (3 × 100 mg/kg, 1 p ) N T P (1 × 2000 mg/kg, p o ) Retrorslne (1 × 35 mg/kg, i p ) Sucrose diet (2 days) Protein-free diet (3 days) Starvatmn (2 days)
311 ± 6 ( P < 0 0 0 1 ) 407±6(P<0001)
Slgntficance c
N-Oxide
d a
95 ~: 7 ( N S ) a
232-k3 (P<0001) a 203 ± 3 ( P < 0 0 0 1 ) a
P<001 P<001
116± 6(NS) a
NS
122 Jz 6 b (P < 0 05) a 178 ± 2 b ( P < 0 0 0 1 ) d 63 ~ 5 b ( P < 0 0 1 )
a
5 0 ± 12b ( P < 0 0 5 ) a 39 ± 2 b (P < 0 001) a 69 ± 3b (P < 0 01) a
a Each result represents the mean of 4 experiments b Retrorsme concentration 0 33 m M Incubation stopped after 60 m m c Probablhtles that differences between rates of formation of pyrrole derivatives and N-oxides are significant a Probablhtles that differences between enzymatic activities in pretreated animals and controls are slgmficant N S , not significant, l p , lntraperltoneally, p o , per os
The effects of substrate concentration were examined to see if differences could be revealed between the enzymatic pathways leading to the formation of pyrrole derivatives and N-oxides The apparent Km values towards retrorsme for the formation of pyrrole derivatives and N-oxides were found to be very stmdar (Table V) Accurate estimations of the Km values with respect to monocrotaline and lasiocarpme N-oxides proved to be impracticable This was due m the case of monocrotahne to the low rate of enzymatic conversion to monocrotallne N-oxide and in the case of laslocarpme, to the low molar extinction coefficient given by its N-oxide, when estimated by the method at present available 1°. When the N-oxides of retrorsine, laslocarpine, monocrotaline or hehotrme, free from the parent alkaloids, were incubated with the mlcrosomal preparations, no conversion to pyrrohc derivatives could be detected The results of a smgle experiment to investigate the conversion of a number of different pyrrohzidme alkaloids to pyrrollc derivatives by a rat-hver microsomal prep° aranon are shown in Table VI. The noncyclic dlester lasiocarpine and the cyclic dlesters having saturated pyrrohzldlne ring structures, platyphylhne and rosmarmine, gave the highest yields of pyrroles, while semisynthetlc short-chain dlesters such as dlacetyl retronecine were amongst those alkaloids giving the lowest yield. Rat-lung microsomal preparations were unable to convert either monocrotahne or retrorsine to pyrrole derivatives Such lung preparations were able to N-demethylate N,N-dImethylanlhne The specific enzymatic activity for this reaction was about 30 % Chem-Btol Interactwns, 3 (1971) 383-396
390
A.R.
MATTOCKS, I. N. H WHITE
TABLE V APPARENT
Kra V A L U E S F O R T H E C O N V E R S I O N OF R E T R O R S I N E ~ M O N O C R O T A L I N E
AND
LASIOCARPINE TO
P Y R R O L E D E R I V A T I V E S A N D T H E C O N V E R S I O N OF R E T R O R S I N E T O N - O X I D E a
Substrate
Km (mM, mean 4. S E ) Pyrrole derivative N-Oxide
RetrorsIne Monocrotahne Laslocarpxne
0 30 4. 0 02 b 0 60 4, 0 06 0 18 4- 0 02
0 32 4. 0 06 b ---
a E a c h value represents the m e a n o f 4 experiments b N o significant d~fference for Km values towards the f o r m a t i o n o f pyrrole derivatives a n d N-oxides f r o m retrorslne
that of hepatic mlcrosomes. When examined by electron microscopy, the mlcrosomal fractions from both organs appeared to be similar. DISCUSSION
The rat-liver enzymes responsible for converting pyrrohzldlne alkaloids to pyrrole derivatives and to N-oxides have their highest specific activities an the mlcrosomal fraction They have many of the characteristics of mlcrosomal mlxed-functlon oxldases 16, which are concerned with the metabohsm of numerous foreign orgamc compounds Conversion of the alkaloids to pyrrole derivatives has previously been shown to take place in liver-tissue slices 4 as well as m the mlcrosomal fraction of rat hver 6'7. The act~wt~es of the mlcrosomal enzymes are influenced by a variety of factors such as sex, age, nutritional status or drug pretreatment of the ammal Iv-19 In the conT A B L E VI A C O M P A R I S O N OF T H E E X T E N T OF E N Z Y M A T I C C O N V E R S I O N O F A N U M B E R O F P Y R R O L I Z I D I N E
ALKALOIDS
T O P Y R R O L E D E R I V A T I V E S BY R A T - L I V E R M I C R O S O M E S In vitro
Alkaloid a
Relative enzymatic activities e (]ormatton of pyrrole derivatives as % of retrorsme)
Acute toxicity LD5o (mg/kg)
Laslocarplne Platyphylhne Rosmarlnlne
260 125 110
88 (ref 3, p 145) N o acute hepatotoxlclty (ref 4) N o acute hepatotoxlclty (ref 4)
Retrorsme Seneclonme Isoseneclomne Anacrotlne
100 100 74 66
38 (ref 3, p 145) 85 (ref 3, p 143)
Monocrotahne Senklrklne Hehotrlne Dmcetyl retroneclne
27 20 19 18
91-175 (ref 3, pp 142, 145) 200-300 (ref 40) 274 (ref 3, p 145) > 900 (ref 29)
Strlgosine b M o n o c r o t a h n e epox~de 7-B-Hydroxy-l-methylene pyrrohzldlne
15 0
N o acute hepatotoxlclty (ref 5)
0
N o acute hepatotoxlclty (ref 25)
a C o n c e n t r a t i o n o f alkaloids was 0 33 m M b C o n c e n t r a t i o n 0.66 m M ¢ Reaction stopped after 60 m l n
Chem-Btol Interactions, 3 (1971) 383-396
CONVERSIONOF ALKALOIDSTO PYRROLEDERIVATIVESAND N-OXIDES
391
version of the pyrrohzldme alkaloids to pyrrole derivatives and to N-oxides, the sex difference with respect to the formation of pyrrole derivatives is greater than that for N-oxides The conversion of retrorsme to pyrrole derivatives and to N-oxides requires oxygen and reduced NADP. N A D H appears to play no direct role in the reaction sequence but in common with many of the mlcrosomal mixed-functlon oxldases has a stablhsmg influence on the enzyme system 1° The usual cations which activate the liver mlcrosomal enzymes, e g. Mg 2+ and Ca z+ (ref 21), stimulate the production of pyrrohc metabohtes from retrorsme It IS of Interest that Mn 2+ potentiates enzymatic activity even in the presence of an optimal concentration of Mg 2+ Thls was found also to be true for Ca 2+ Since chemically reduced N A D P H was employed, the action of these ions cannot be on the N A D P H generating system Ca 2+ did not further activate the enzyme when Mn 2+ was also present, so both might be acting at similar sites in the mlcrosomal complex.
Inhlbltors A number of compounds which inhibit liver mlcrosomal drug-metabohsmg enzymes, such as SKF 525A and carbon monoxide, also inhibit the formation of pyrrohc and N-oxide metabohtes Inhibition of the former is significantly greater than that of the latter when carbon monoxide is used Mlcrosomal systems responsible for N-oxidation do not always behave in this way For example, the formation of N,N-dlmethylamhne-N-oxlde from N,N-dlmethylanlhne by pork-hver mlcrosomes is little affected by SKF 525A or carbon monoxide 2z Nicotlnamlde was at one time routinely included m incubation mixtures by many workers investigating mlcrosomal drug metabolism to inhibit pyridme nucleotadases 23. In our experiments, nlcotmamlde consistently inhibited the formation of pyrrole derivatives but had little effect on the production of N-oxldes The action of mcotmamide on the mlcrosomal enzymes is complex For some reactions It acts as a competitive or a mixed type of inhibitor 24, while for others it enhances enzymatic activity25.
Mlcrosomes from pretreated rats Hepatic mlcrosomes from rats pretreated with phenobarbitone converted retrorsine much more rapidly to pyrrole metabohtes than the corresponding control animals The formation of N-oxide was increased to a smaller extent These results were obtained whether phenobarbltone was administered by mtraperltoneal injection or given as a solution in the drinking water The action of DDT was similar to that of phenobarbitone, the formation of pyrrole derivatives being induced to a greater extent than the formation of N-oxides In contrast to D D T and phenobarbltone, the polycychc hydrocarbon 3-methylcholanthrene, which is also capable of inducing a number of hepatic drug-metabollslng enzymes 17, failed to significantly enhance the production of either pyrrohc or N-oxide metabohtes The amounts of pyrrohc metabolltes formed from retrorslne were decreased when mlcrosomes were prepared from rats which had been starved or fed a sucrose or protein-free diet. These findings are consistent with the results of other workers
Chem-Btol Interactions, 3 (1971) 383-396
392
A R MATTOCKS, I. N. H. WHITE
who have shown that protein-depletion of rats leads to a reductmn in the hver mlcrosomal drug-metabohsing enzymes Is. The acute toxicity of retrorslne in vtvo was also decreased in rats fed on a sucrose diet 26, a result which is compatible with the view that acute toxicity is related to the microsomal metabolism of this alkaloid Amlnoacetonitrlle has been reported as offering partial protection against the acute necrotic effect m livers of rats following administration ofdImethylnItrosamlne 27 The action of amlnoacetonltrde was investigated to see if it could reduce the formation of pyrrole derivatives and thereby possibly protect the animal against the acute hepatotoxtc effects of the pyrrohzldlne alkaloids. However, the latter seems unlikely since pretreatment with th~s compound slightly actwated the mlcrosomal system. N T P IS a mixture of aryl phosphates which inhibit esterase activity 28. Rats were pretreated with this material to assess the importance of liver esterases in the metabolism of the pyrrollzidlne alkaloids The result was a significant increase m the microsomal metabohsm of retrorsine to pyrrole derivatives The toxicity to rats of diacetyl retronecme which is very easily hydrolysed was increased after treatment with N T P 29. However, it is not certain whether these results were due to esterase inhibition or to mductmn of the mlcrosomal enzymes by some constituents of the N T P mixture. It has been proposed that the initial pyrrollc metabohtes from the hepatotoxlc alkaloids might act as blfunctional alkylatlng agents 4's. Since these could react with nucleophlhc groups In proteins, the mlcrosomal enzymes themselves might be attacked Pretreatment of rats with retrorslnejust below an LDso dose brought about a decrease in the subsequent production of pyrrole by the isolated mxcrosomes m vitro The significance of this result is difficult to assess, since some hepatic cell necrosis had probably occurred, but it is evident that 20 h after a near-lethal dose ofretrorslne the system retains a high capacity for producing pyrrohc metabohtes M e t a b o h s m o f other alkaloids
A number of pyrrohzldlne alkaloids are converted into pyrrole derivatives by rat-hver microsomal enzymes Two d~fferent types of pyrrohc metabohtes have been identified Alkaloids which are esters of unsaturated amino alcohols, including laslocarplne, hehotrlne, retrorslne, seneclonlne, anacrotme, monocrotahne, senkirklne, and the semlsynthetic compounds lsoseneclonlne and dlacetylretronecme, give dlhydropyrrohzlne esters which are more reactive than the parent alkaloids and are capable of acting as alkylating agents 4's'7. As prewously reported, the alkaloids platyphylhne 5 and rosmarmlne% which have saturated pyrrohzldlne rings, gave relatively large amounts of pyrrohc derivatives. We have evidence that in rosmarlnme and platyphylhne ring A is dehydrogenated giving pyrrole derivatives which are quite different from those from the hepatotomc alkaloids (Fig. 2) (ref. 26). Both the ester groups in I are highly labile due to conjugation with the nitrogen, whereas the ester II xs relatively unreactive Platyphylhne and rosmarinlne have not been shown to be hepatotoxlc. Platyphylhne does not produce acute liver damage in the rat 3°'31, and has been used chnlcally as a substitute for atropine without causing any long-term hepatic or renal damage 32. Chem-Btol Interactions, 3 (1971) 383-396
CONVERSIONOFALKALOIDSTO PYRROLEDERIVATIVESANDN-OXIDES I
OCO
I
~
393
I
OCO
(~ I COOH I-
CO0
R
l
CH2OCO
L
R
l
CH2OCO
(b)
Fig 2 Conversxonto pyrrohc metabohtes of (a) toxic pyrrohzldme alkaloxds (such as seneclonme) and (b) nontoxic alkaloids (such as rosmarmme)
All the alkaloids which are known to be hepatotoxlc form pyrrole derivatives of the first, reactive type W~thm this group, there is some relaUonshlp between amounts of pyrrohc metabohtes formed m vitro from the alkaloids and their acute hepatotoxlcit]es to the rat m vtvo, although the latter are taken from several sources and may not necessarily be accurate for our strata of rats Thus retrorsme, with high toxicity, gwes a large amount of pyrrolic metabohtes, whereas hehotrme, which has low toxicity, gives much less There are exceptions, and the correlations are better when hver metabohte levels are measured tn vtvo 26. Lasiocarpme has about half the acute tOXlClty of retrorslne when measured m vtvo (ref. 3, p. 145), whereas the amount of pyrrole formed m vttro is more than double that from retrorsme. Laslocarpme has also been shown to give consistently stronger pyrrole colour than retrorsme m rat-hver tissue shces 4 and in human-embryo hver-txssue shces 33 However, the amounts ofpyrrole m rat hver 2 h after an LDso dose of las10carpme or retrorsme are similar 26 It therefore seems hkely that some factor other than mlcrosomal enzymatic activity limits the rate of pyrrole formation from laslocarpme m vtvo. One possibility is that the noncychc dlester laslocarpme is more susceptible to attack by hver esterases m vtvo and this reaction competes with the production ofpyrrole denvattves Seneclomne, which differs from retrorsme by one hydroxyl group, gives as much pyrrole as retrorsine but is less toxtc. The effect of a change in the stereochemistry of the acid moiety is seen by comparing seneclonlne and lsoseneclonlne, which are esters of retronecine with (-r) and (--) seneclc acid, respectively 26 Stngosme is a saturated alkaloid 34 of which too little was available for further study. T o x t c z t y to l u n g s
Monocrotahne and fulvlne, when administered to rats m vtvo at acute LDso doses, cause lung lesions m addition to their hepatotoxxc actions 2,as. Retrorsme generally has no effect on lung tissue As previously reported 29, the conversion of e~ther monocrotahne or retrorsme to pyrrole derivatives could not be demonstrated using lung microsomal preparations. The inability of human-embryo lung-tissue slices Chem-Blol Interacttons,
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A R MATTOCKS, I N H. WHITE
to convert laslocarplne, fulvlne and retrorsme to pyrroles has also been reported 33. Synthetic pyrrohc esters can cause lung damage similar to that produced by monocrotahne and fulv]ne 29'36, and it is likely that some metabohtes formed from these alkaloids in the liver are sufficiently stable to escape Into the circulation and cause lung lesions Metabohc pathways The apparent Km values for formation of pyrrole derivatives and N-oxides in rat-hver mlcrosomal preparations are similar, but this does not indicate whether the two metabohtes are formed independently or if the N-oxide Is an intermediate in the formation of pyrrole derivatives Although rat-liver slices incubated m vitro produce small amounts of pyrrole derivatives from the N-oxides 4, this is not demonstrable using the isolated mlcrosomal enzyme system This finding is in agreement with the results of other workers 7 Values of Km towards retrorslne pyrrole and N-oxlde formation are 6-7 times higher than values obtamed with respect to the hydroxylatlon of small aromatic molecules, e g. aniline, but are of the same order as those for reactions involving more complex compounds, e g the N-demethylatlon of ethylmorphlne 37. Neither the pyrrollc or N-oxide pathways are greatly affected by pretreatment of rats with 3-methylcholanthrene Both are mduced by pretreatment with phenobarbltone or D D T , but the induction of pyrrole formation is greater than that of N-oxide Nlcotlnamlde has an inhibitory effect only on pyrrole formatton Carbon monoxide inhibits the formation of pyrrole derivatives more than that of N-oxides While not conclusive, the results favour the view, shared by other workers 7, that pyrrohc metabohtes are formed from the alkaloids through a separate enzymatic pathway and not through the N-oxides. Possible reaction mechamsms The formation of pyrrohc derivatives means that the pyrrohzldme ring is dehydrogenated The mechanism of the dehydrogenation brought about by rat-hver fractions is not known, but it may be pointed out that this need not be a new type H
5
3 H
I
CH3 II
III
IV
Fig 3 Hypothetical conversion o f u n s a t u r a t e d pyrrohzldlne alkaloids to dlhydropyrrollzlnes (IV), initiated by enzymatic hydroxylatlon (of I) or d e m e t h y l a t l o n (of II) F o r slrnpllClty, ring substltuents n o t directly involved m the reactions have been omitted
Chem -Btol Interactions, 3 (1971) 383-396
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVESAND N-OXIDES
395
o f m l c r o s o m a l reaction R e a c ti o n s c o m m o n l y catalysed by m l c r o s o m a l enzymes, such as N - o x l d a t m n or, m o r e p r o b a b ly , C-hydroxylatton, could be the m l t l at l n g process E n z y m a t i c h y d r o x y l a t l o n at positions 3 or 8 o f the d e h y d r o p y r r o l l z l d l n e ring (F~g. 3, I) should lead to the d l h y d r o p y r r o l l z l n e structure (IV) by a s p o n t a n e o u s sequence o f chemical (nonenzymatlc) reactions. E n z y m a t i c N - d e m e t h y l a t l o n o f alkaloids h a v m g o t o n e c l n e - t y p e bases (II) should lead to (III) which again w o u l d s p o n t a n e o u s l y give (IV). Wh ereas the p y r r o l m d l n e N-oxides are readily d e h y d r a t e d by heat or chemical r e a c t m n s 6'38'39 to pyrroles hke IV (Fig. 3), this reaction is n o t b r o u g h t a b o u t by the m l c r o s o m a l e n z y m e system, and it appears likely that these c o m p o u n d s are 'detOXlCatmn p r o d u c t s ' which, being highly water soluble, are rapidly excreted. F o r m a t i o n o f the different pyrroles (II, Fig 2) f r o m r o s m a r l n l n e an d platyp h y l h n e p r o b a b l y depends u p o n loss o f the car b o x y l at e a m o n at C-7 as p a r t o f a r e a c t m n sequence initiated by enzymatic h y d r o x y l a t l o n in rlng A. ACKNOWLEDGEMENTS W e t h a n k D r D. H G CROUT (Exeter University) for specimens o f senklrkme -J
/
and h y d r o x y s e n k l r k l n e ; P r o f F. SANTAW ( O l o m o u c ) f o r p l a t y p h y l h n e , Dr. W. H BUTLER for electron m l c r o g r a p h s o f t~ssue f r a c t m n s , and T HOUGEN and B. A J ALEXANDER for skilled technical assistance. REFERENCES 1 J DAVIDSON,The action of retrorsme on rat's hver, J Pathol Bactertol, 40 (1935) 285-295 2 R SCHOENrAL,Toxicology and carcmogemc action of pyrrohzldme alkalmds, Cancer Res, 28 (1968) 2237-2246 3 L B BULL, C C J CULVENORAYD A J DICK, The Pyrrohzldme Alkaloids Their Chemistry, Pathogemclty and Other Biological Properties, North-Holland, Amsterdam, 1968 4 A R MATTOCKS,Toxlclty of pyrrolmdme alkaloids, Nature, 217 (1968) 723-728 5 C C J Cr.rLVENOR,D T DOWNING,J A EDGARAND M V JAGO,Pyrrohzldlne alkaloids as alkylatmg and antxmltotlc agents, Ann N Y Acad Set, 163 (1969) 837-847 6 A R MATTOCKS,Iron (II)-catalysed conversion of unsaturated pyrrollzldme alkaloid N-oxides to pyrrole derivatives, Nature, 219 (1968) 480 7 M V JAGO,J A EDGAR,L W SMITHAND C C J CULVENOR,Metabohc eonversmn ofhehotrtdme-based pyrrohzldme alkaloids to dehydrohehotndme, Mol Pharmaeol, 6 (1970) 402-406 8 W N ALDRIDGE,R C EMERYAND B W STREET,A tissue homogemser, Btoehem J , 77 (1960) 326-327 9 O H LowRY, N J ROSEBROUGH,A L FARRAND R J RANDALL,Protein measurement with the Fohn phenol reagent, J Btol Chem, 193 (1951) 265-275 10 A R MATTOCKSAND I N H WroTE, EstlmaUon of metabohtes of pyrrohmdme alkaloids m ammal t~ssues, Anal Bmchem, 38 (1970) 529-535 11 M DIXONAND E C WESB, Enzymes, Academic Press, New York, 1964, p 69 12 N E GooD, G D WINGET,W WINTER,T N CONNOLLY, S IZAWAAND R M M SINGH, Hydrogen mn buffers for biological research, Biochemistry, 5 (1966) 467-477 13 H M BRUCEaND A S PARKES,Letter to Editor, J Ammal Tech Ass, 7 (1956) 54 14 W J MARSHALLAND A E M MCLEAN,The effects of oral phenobarbltone on hepatic microsomal cytochrome P-450 and demethylatlon activity in rats fed normal and low-protein dxets, Btochem Pharmaeol, 18 (1969) 153-157 15 A E M MCLEANAYD E K MCLEAN,The effect of diet and l,l,l-tnchloro-2,2-bls-(p-chlorophenyl)ethane (DDT) on mlcrosomal hydroxylatmg enzymes and on sensmwty of rats to carbon tetrachlonde poisoning, Bmchem J , 100 (1966) 564-571 Chem-Btol Interactions, 3 (1971) 383-396
396 16 17 18 19
20
21 22 23 24 25
26 27
28 29 30 31 32 33 34 35 36 37
38 39
40
A . R . MATTOCKS, I. N. H WHITE H S MASON,J C NORTH AND M VANNESTE,M l c r o s o m a l mixed-function oxidations the metabo h s m o f xenoblotlcs, Federation Proc, 24 (1965) 1172-1180 A H CONNEY AND J J BURNS, Factors affecting drug m e t a b o h s m , In S GARATTINI AND P A SHORE ( E d s ) , Advances in Pharmacology, Vol 1, A c a d e m i c Press, N e w York, 1962, pp 31-57. G P QUINN, J AXELROD AND B B BRODIE, Species, strata a n d sex differences in m e t a b o l i s m o f h e x o b a r b l t o n e , amldopyrene, antlpyrene a n d aniline, Biochem Pharmacol, 1 (1958) 152-159 R L FURNER, T E GRAM AND R E STITZEL, T h e influence o f age, sex a n d drug t r e a t m e n t on m l c r o s o m a l drug m e t a b o l i s m in four rat strains, Btochem Pharmacol, 18 (1969) 1635-1641 R W ESTABROOKAND B COHEN, Organtsatton o f the m l c r o s o m a l electron t r a n s p o r t s y s t e m , in J R GILLETTE,A H CONNEY,G J COSMIDES,R W ESTABROOK, J R FOUTS AND G J MANNERING ( E d s ) , Mtcrosomes and Drug Oxidations, A c a d e m i c Press, N e w York, 1969, p 102 M A PETERS AND J R FOUTS, T h e influence o f m a g n e s i u m a n d s o m e other divalent cations on hepatic mlcrosomal drug m e t a b o l i s m m vitro, Btochem Pharmacol, 19 (1970) 533-544 D M ZIEGLER AND F H PETTIT, Mlcrosomal oxldases, I T h e isolation a n d dlalkylarylamlne o x y g e n a s e activity o f pork liver mlcrosomes, Biochemistry, 5 (1966) 2932-2938 P J G MANN AND S H QUASTEL,Nlcottnamlde, cozymase a n d tissue m e t a b o l i s m , Btochem J , 35 (1941) 502-517 J B SCHENKMAN,J A BULL AND R W ESTABROOK,O n the use o f n l c o t m a m l d e m assays for m l c r o s o m a l mixed-function oxIdase activity, Btochem Pharmacol, 16 (1967) 1071-1081 A NEUGEBAURER,F K SPLINTER, D HAFKE, R KOBER, H SCHIRLITZ AND W KLINGER, Kinetische U n t e r s u c h u n g e n uber die Enzymatlsche H y d r o x y h e r u n g v o n P h e n a z o n , oxidative O - d e m e t h y h e r u n g von K o d e m u n d N-demethyllerung von A m m o p h e n a z o n durch Rattenleberm l c r o s o m e n , Btochem Pharmacol, 16 (1967) 1559-1576 A R MATTOCKS, u n p u b l i s h e d results L FIUME, G CAPADELLI-FIUME,P N MAGEE AND J HOLSMAN,Cellular injury a n d carcinogenesis Inhibition o f m e t a b o l i s m o f d i m e t h y l m t r o s a m I n e by amtnoacetomtrlle, Btochem J , 120 (1970) 601-605 H F BONDY,E J FIELD, A N WORDEN AND J P W HUGHES,A study on the acute toxicity o f the trl-aryl p h o s p h a t e s used as plasticlzers, Brtt J Ind M e d , 17 (1960) 190-200 A R MATTOCKS, T h e role o f acid moieties in the toxic actions o f pyrrohzldxne alkaloids o n hver a n d lung, Nature, 228 (1970) 174 R SCHOENTAL,Hepatotoxlc action o f p y r r o h z l d m e (Senecto) alkaloids in relation to their structure, Nature, 179 (1957) 361-363 C C J CULVENOR,A T DANN AND A T DICK, Alkylatlon as the m e c h a n i s m by which the hepatotoxlc pyrrohzldine alkaloids act on cell nuclei, Nature, 195 (1962) 570-573 F L WARREN,T h e pyrrohzldlne alkaloids, in L ZECHMEISTER ( E d ) , Progress m the Chemistry of Organic Natural Products, Vol 12, Springer, Vienna, 1955, p 259 S J ARMSTRONG AND A J ZUCKERMAN,Production o f pyrroles from pyrrollzldme alkaloids by h u m a n e m b r y o tissue, Nature, 228 (1970) 569 A R MATTOCKS, Strlgosine, the m a j o r alkaloid o f Hehotroptum strlgosum, J Chem Soc , (1964), 1974-1977 J M BARNES,P N MAGEE AND R SCHOENTAL,Lesions in the lungs a n d livers o f rats p o i s o n e d with the p y r r o h z l d m e alkaloid fulvine a n d its N-oxide, J Pathol Bactertol, 88 (1964) 521-531 W H BUTLER, A R MATTOCKS AND J M BARNES, Lesions m the lungs a n d livers o f rats given pyrrole derivatives o f pyrrohzldlne alkaloids, J Pathol, 100 (1970) 169-175 T E GRAM, A M GUARINO, D H SCHROEDER AND J R GILLETTE,C h a n g e s m certain kinetic properties o f hepatic m i c r o s o m a l a n l h n e hydroxylase a n d e t h y l m o r p h l n e demethylase associated with p o s t n a t a l d e v e l o p m e n t a n d m a t u r a t i o n in male rats, Blochem J , 113 (1969) 681-685 A R MATTOCKS, D l h y d r o p y r r o h z l n e derivatives from u n s a t u r a t e d pyrrohzldine alkaloids, J Chem Soc C, (1969)1155-1162 C C J CULVENOR, J A EDGAR, L W SMITH AND J H TWEEDDALE, D i h y d r o - p y r r o h z i n e s , III Preparation a n d reactions o f denvaUves related to the pyrrohzldlne alkaloids, Austrahan J Chem, 23 (1970) 1853-1867 R SCHOENTAL,Hepatotoxlc activity o f retrorslne, s e n k I r k m e a n d hydroxysenkirkIne In newbol:n rats, a n d the role o f epoxldes in carcinogenesis by p y r r o h z l d m e alkaloids a n d aflatoxms, Nature, 227 (1970) 401-402
Chem -Btol Interactions, 3 (1971) 383-396
383
THE C O N V E R S I O N OF P Y R R O L I Z I D I N E A L K A L O I D S TO N-OXIDES A N D TO D I H Y D R O P Y R R O L I Z I N E D E R I V A T I V E S BY R A T - L I V E R M I C R O S O M E S IN VITRO
A R MATTOCKS AND I N H WHITE
M R C Toxtcology Umt, Medtcal Research Counctl Laboratories, Woodmansterne Road, Carshalton, Surrey (Great Brltatn) (Received January 5th, 1971) (Rewslon accepted February 4th, 1971)
SUMMARY
The enzymatic conversion of pyrrohzldlne alkaloids to dlhydropyrrolazlne derivatives ('pyrrole derivatives') and to N-oxides has been investigated. In rat liver both reactions were catalysed by an enzyme present in the mlcrosomal fraction, typical of the mixed-function oxldases Thus the conversion of alkaloids to pyrrole derivatives and to N-oxides required oxygen and reduced N A D P and was activated by Mg 2+, Mn 2+ or Ca 2+ at concentrations of 5 10 -3 M The liver mlcrosomal system was less active an the female rat than an the male and was inhibited by carbon monoxide and by SKF 525A The conversion of retrorslne to both pyrrohc metabohtes and to N-oxides was significantly reduced by pretreatment with phenobarbltone or DDT, but the Induction of pyrrole derlvatwes was greater than that of N-oxide The conversion of retrorslne to pyrrole derivatives was lower an liver mlcrosomal fractions from rats which were starved or fed on protein-free d~ets Differences between the rates of formation of pyrrole derivatives and N-oxides followed the ancluslon of nlcotmamlde m the incubation mixtures containing macrosomes from untreated rats, or substitution of a carbon monoxide-oxygen mixture for air during the incubation period The Km values towards the formation of pyrrolic denvatwes and N-oxides from retrorslne from normal rat-laver mlcrosomes were similar No conclusive evidence was found to establish whether N-oxides were intermediate m the formation of pyrrole derivatives. However, pyrrole denvatwes could not be detected when N-oxides were added to the hepatic mlcrosomal system m vztro Rat-hver mlcrosomal enzymes converted a number of alkaloads havang either 1,2-dehydropyrrohzadlne or saturated pyrrohzldlne ring structures to pyrrole denvatwes The pyrrole derlvatwes from these two groups of alkaloids had different chemical properties Only alkaloads in the first group were hepatotoxlc, and in general the more toxic alkaloids in th~s group were Abbreviation NTP, nontoxic plasticizer
Chem-Blol Interacttons, 3 (1971) 383-396
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A R. MATTOCKS, I N H WHITE
those from which the greatest amounts of pyrrole derivatives were produced m vitro, although there were exceptions The enzymatic conversion of retrorsme or monocrotahne to pyrrohc derivatives could not be demonstrated in lung mlcrosomal fractions
INTRODUCTION
The action of many pyrrohzldme alkaloids in bringing about both acute and long-term changes in the livers of experimental animals has been described 1'2. These alkaloids are metabohsed by a variety of pathways such as hydrolysis, hydroxylatlon and N-oxidation (ref. 3, p. 215). Alkaloids having a 1,2-dehydropyrrohzldlne ring structure undergo conversion in the liver to pyrrole-hke derivatives *'5 and it has been suggested that such reactive derivatives may be responsible for toxic effects4 . The hepatic microsomal system has been imphcated in this reaction 6 and the conversion of hehotrldlne-based alkaloids to dlhydropyrrohzine derivatives m vitro has recently been demonstrated using rat hepatic mlcrosomal preparations 7. The work to be described examines some of the propemes of the enzymes in rat hver which are responsible for converting the alkaloids to N-oxides and pyrrole derivatives The efficiency of the system m the productmn of pyrrole derwatwes from a number of dlfferent pyrrohzidme alkaloids m vitro is compared with the published acute tOXlCmes of the alkaloids tn vtvo The effects of pretreatmg rats in a number of ways, on the mlcrosomal metabolism of these alkaloids m vitro are also described. MATERIALS AND METHODS
Chemicals were obtained from B D H L t d , unless otherwise mentmned N A D , NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Boehrlnger Corporation (London) Ltd Preparation of hver fractions
Albino Porton rats (150-200 g, males except where otherwise stated) were killed by a blow on the head and exsangumated, normally between 10 00 and 11 00 h. The hvers were quickly removed and placed in ice-cold 1 15 ~ KC1. All the remaining procedures were carried out at 0-4 ° unless otherwise indicated. After being blotted, the livers were weighed, minced with scissors and 33 ~ (w/v) liver homogenates prepared in 1.I 5 ~ KC1 using a pestle-type homogemser with 0 02-inch clearance 8. The homogenate was centrifuged at 12 000 g for 20 m m in a MSE HighSpeed 18 centrifuge. The precipitate was resuspended m 1 15 ~ KC1 equal in volume to that of the original homogenate, while the supernatant was further centrifuged at 105 000 g for 60 mln in a Splnco Model L ultracentrifuge to give a mlcrosomal pellet and a supernatant (soluble) fraction. When washed mlcrosomes were required, the pellet was resuspended in the original volume of 1.15 ~ KC1 and resedlmented as before The mlcrosomal pellet was finally resuspended in a volume of 1 15 ~ KC1 Chem-Btol Interactions, 3 (1971) 383-396
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVES AND N-OXIDES
385
such that 1 ml was equivalent to 1 g of hver wet weight. Mlcrosomal protein was estimated by the method of LOWRY et al 9 with bovine serum albumin as the protein standard
Preparation of lung microsomal fractton Rats were decapitated and thoroughly exsangulnated, and the lungs from 6-8 animals were removed as quickly as possible and pooled in ice-cold 1 15 ~ KC1. After being homogenised in the same way as the hver, the suspensmn was filtered through a single layer of surgical gauze. Subsequent procedures for cell fractIonatlon were carried out in a manner identical to those already described for rat liver
Assay procedures Incubation mixtures of 3 ml final volume consisted of 0 25 M phosphate buffer (pH 7 4), 14 m M MgC12, 0 25 m M N A D , 0 22 m M N A D P , 4.0 m M glucose-6-phosphate and 0.5 I.U./ml glucose-6-phosphate dehydrogenase Substrates were pure crystalhne pyrrohzldine alkaloids neutrahsed wlth an equivalent of HC1 or pyrrohzldme N-oxides at concentrations of 3 3 mM, unless otherwise mdxcated. Following equlhbratton at 37 °, reactions were started by the a d d m o n of 0 5 ml of the mtcrosomal suspensxon (containing about 8 mg mlcrosomal protein) Flasks were shaken at 100 strokes per mln in air for 10 m m unless otherwise indicated Reactions were stopped with ethanol N-Oxide metabohtes were chemically converted to pyrrole derivatives which were then estimated using Ehrhch reagent. Pyrrohc metabohtes were estimated directly by an Ehrhch reactmn Detads of these procedures have been described ~°.
Determmatton of Km values Apparent K,, constants were determined by means of Woolf plots (see ref 11) ([S]/v vs [S]), best-fitting lines being drawn by the method of least squares A range of alkaloid concentrations from 0 1 m M to 1 m M was employed.
Effect of mhtbttors The action of a number of lnhlbltors was investigated by adding them to the reaction mixture immediately prior to incubation. Compounds tested included fldlethylamlnoethyl dlphenylpropylacetate (SKF 525A; Smith, Klyne and French L t d ) (5. 10 -4 M), K C N (5 10 -3 M), and nlcotmamide ( 1 . 4 . 1 0 -2 M) In experiments where air was not employed as the gas phase, the appropriate gas mixture was passed through flasks containing the reaction mixture and mlcrosomes but no alkaloid, at 4 ° After 5 mln, the flasks were equlhbrated at 37 ° and the reaction started by the addmon of alkaloid A gas flow of 500 ml/mln through the flasks was maintained for the duration of the experiment.
The effect of cations The actions of the chlorides of Mg z+, Mn 2+, Ca z÷, Co 2÷ and Cu 2÷ were tested by their Inclusion m the reaction mixtures at concentrations of 5 10 -4 M and 5 • 10 -3 M Procedures were the same as described above Washed mlcrosomes were used. Incubatmn mixtures contained 0 25 M Hepes (N-2-hydroxyethylpxperChem-Btol Interactions, 3 (1971) 383-396
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A. R. MATTOCKS, I N. H. WHITE
azme-N-2-ethanesulphonlc acxd) (Sigma Chemxcal Co ) buffer 12, 3 3 m M retrorslne, 1 m M N A D P H (chemically reduced), 0.25 m M N A D and 1 15 % KCI to give a final reactmn volume of 3 ml
Diets and predosmg Rats were normally fed ad hb. a M R C 41B diet 13 with free access to water at all times. Sodium phenobarbitone (100 mg/kg) was administered either by lntraperitoneal rejection in saline, 72, 48 and 24 h before the rats were killed, or as a 0.1% solution in the drinking water for 7 days 14. The followmg compounds were administered by lntraperltoneal rejection at the indicated times before the rats were killed: 3-methylcholanthrene (Koch-Light Laboratories), 20 mg/kg in trioctanoin (Eastman) at 72, 48 and 24 h; 1,1,1-mchloro-2,2-bls(p-chlorophenyl)ethane (DDT) (Geigy), 75 mg/kg in trioctanoxn at 72 h; amlnoacetonltrde blsulphate (Koch-Light Laboratories), 100 mg/kg m sahne 48, 24 and 1 h; and retrorsme hydrochlonde, 35 mg/kg in saline at 20 h NTP*, 2000 mg/kg, was administered orally as a 50 % solutmn in arachls oil, 60 h before the rats were killed. Rats were also maintained on diets of" Analar granular sucrose for 2 days; on the protein-free diet described by MCLEAN AND MCLEAN ~5 for 3 days; or starved for 2 days before being killed. RESULTS
Table I shows a comparison between enzymatic activities associated with the conversion of retrorslne to pyrrole derivatives and to N-oxides in subcellular fractions of rat liver The recoveries, expressed as percentages of the activities found in the original liver homogenate, appeared to be similar for both reactions. The highest specific activity for the formation of both pyrrole derivatives and N-oxides was In the mlcrosomal fraction Specific mlcrosomal activities, for the formation of pyrrole derivatives and N-oxides in female rats were respectively about 15 % and 30% those of male rats. The time-courses for the enzymatic conversion of retrorslne to pyrrole denvattves and to N-oxides by a male rat-hver mlcrosomal fraction are shown in Fig. 1. The rates of conversion to both metabolites remained linear for the first 10 mln when both N A D P H and N A D H were present. Omission of N A D H had little effect on the initial enzymatic activities, but both reactions terminated more quickly than when both cofactors were present. No enzymatic conversion of retrorslne to pyrrole derivatives or to N-oxides could be detected in the presence of N A D H alone. A number of cations were examined for their effects on the mlcrosomal conversion of retrorsine to pyrrohc derivatives (Table II). Apart from Cu z÷ which was strongly inhibitory, the remaining cations tested had little effect at concentrations of 5 • 10 . 4 M At a concentration of 5. 10 -a M, however, Mg 2+, Mn 2+ and Ca 2+ all stimulated microsomal activity Experiments designed to investigate the effects of one cation In the presence of another, indicated that in the presence of Mg 2+ either Mn 2+ or Ca 2+ caused further activation, but Mn 2+ and Ca 2+ together did • A mixture o f tn-aryl p h o s p h a t e s (Lot N o N r Chesterfield, U K )
Chem-Blol Interactions, 3 (1971) 383-396
S 1853, C o a h t e Chemical Products L t d , Bolsover,
387
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVES AND N-OXIDES TABLE I RECOVERY
AND
SPECIFIC ENZYMATIC
ACTIVITIES ASSOCIATED WITH
TO PYRROLE DERIVATIVES OR TO N-OXIDES
Fractton
IN VARIOUS SUBCELLULAR
Specific acttvlty (nmoles/mm/mg protein 4- S E ) Pyrrole dertvattve
N- Oxtde
1 06 4- 0 16
0 83 4- 0 04
0 51 1 59 3 20 0 51
0 67 1 14 2 27 0 40
THE
CONVERSION
OF RETRORSINE
FRACTIONS OF RAT LIVER a
Recovery ( % o f mean homogenate acttvtty 4- S E ) Pyrrole N- Oxide derlvattve
Male rats Homogenate Cell debris, nuclei, mltochondrm, etc 12 000 g supernatant Mlcrosomes 105 000 g supernatant
4± 44-
0 0 0 0
10 12 40 15
4444-
0 0 0 0
05 12 19 10
344-2 65 4- 5 33 4- 3 13+1
31 72 34 11
:t:2 b 4- 5b 4- 4 b -{- 1b
Female rats Mlcrosomes
0 54 4- 0 12
0 72 4- 0 07
a Each value represents the mean o f 4 experiments b NO slgmficant difference between the percentage recovery of pyrrole derivative and N-oxide
not activate the system to a slgmficantly greater extent than either 1on by itself. Some compounds known to be mhlbltors of the mlcrosomal drug-metabohslng system were examined for their effects on the metabohsm of retrorsme to pyrrole derivatives and to N-oxides using a rat-hver mlcrosomal preparation (Table III) Incubanons carried out anaerobically, m the presence of SKF 525A, or a carbon monoxide + oxygen mlxture, all resulted m a slgmficant decrease in the amount of pyrrole and N-oxide formed. Substltunon of 100 % oxygen for mr had little effect on the production of pyrrohc metabohtes but significantly stimulated N-oxide formation when measured over o Q.
~ o
~0c
0 ~b
_~ 8C
g E
,o
60
,.0
40
[]
20
o
[]
J
I0
20
.3b 4o 5'0 INCUBATION TIME (MIN)
6~0
Fig 1 Time-course o f the rat hepatic mlcrosomal conversion of retrorsme to pyrrole denvataves and to N-oxides in wtro Pyrrole denvanves produced in the presence o f N A D P H and N A D H (O) m the presence o f N A D P H ([5]). N-Oxade produced m the presence o f N A D P H and N A D H ( O ) , in the presence o f N A D P H (11)
Chem-Btol Interactlons, 3 (1971) 383-396
388
A. R. MATTOCKS, I N. H. WHITE
T A B L E II EFFECT OF VARIOUS CATIONS ON RAT-LIVER MICROSOMAL CONVERSION OF RETRORSINE TO PYRROLIC METABOLITES In vltro a
Enzyme activity ( ~ o f mean control values 4- S E ) at cation concn ( M )
Cation
M g 2+ M n 2+ Ca 2+ Co z+ Cu 2+ MgZ+ + Mn2+ b Mg2+ + Ca2+ b Mg2+ q_ Ca2+ 4- Mn2+ b
5 I0 - 4
5
103 102 104 108 29
115 127 138 70 20 120 127 132
4- 5 4- 1 4- 4 4- 2 4-4
I0 - 3 44444444-
5 15 1 1 1 4 1 4
a Each result represents the m e a n of 2 experiments b MgZ+ c o n c e n t r a t i o n was 1 10 - 2 M
a 10-mm penod. Potassium cyamde proved to have little effect on either system. Nlcotlnamlde was without effect on the conversion of retrorsme to N-oxide but brought about a significant lnhlbttmn of the formation of pyrrohc derivatives Table IV shows the effect of pretreatmg rats with a number of compounds Phenobarbltone and D D T pretreatment both greatly stimulated the hver mlcrosomal conversion of retrorsme to pyrr.ole denvatlves in vitro, while the effect on N-oxide productmn was somewhat less pronounced Pretreatment of rats with ammoacetomtrlle stimulated the productmn of pyrrohc derivatives from retrorsme. NTP, an organophosphorus esterase mhxbltor, also stimulated the formation of pyrrohc metabohtes Retrorslne pretreatment at shghtly less than the acute LDso dose brought about a marked decrease m the hver mlcrosomal pyrrole formatmn The results of experiments using protein-depleted rats showed that lrrespect~ve of the way m which this was achieved, protein depletmn brought about a slgmficant decrease in the hver m~crosomal conversmn of retrorsme to pyrrole derlvatwes. T A B L E III THE EFFECT OF VARIOUS GAS MIXTURES AND INHIB1TORS ON THE In vitro CONVERSION OF RETRORSINE TO PYRROLE DERIVATIVES AND TO N-OXIDES BY RAT-LIVER MICROSOMAL PREPARATIONSa
Compound
Enzyme activity ( % o f mean control values 4- S E ) Pyrrole derivatives
N i t r o g e n (100K) C a r b o n m o n o x i d e -k o xygen (80 20) Oxygen ( 1 0 0 ~ ) S K F 5 2 5 A ( 5 10 - 4 M ) P o t a s s i u m cyanide (5 1 0 - 3 M ) NlCOtlnamlde (1 4 1 0 - 2 M )
20 4- 1 (P < 0 001) c 42 102 35 100 73
4- 3 (P < 0 001) c 4- 4 (N S )c 4-4(P<0001) c 4- 2 (N S )c 4- 2 (P < 0 001) e
Stgmficance b
N-Oxide 22 4- 1 ( P < 0 0 0 1 )
e
68±2(P<0001) e 114 4- 2 (P < 0 01) ¢ 41 4 - 2 ( P < 0 0 0 1 ) e 90 4 - 5 ( N S ) c 98 4- 2 (N S ) c
NS P <001 N S NS NS P < 001
a E a c h result represents the m e a n o f 4 experiments b Pro babilities t h a t differences between rates o f f o r m a t i o n o f p y r r o l e derivatives a n d N-oxides are significant N S , n o t significant c P r o b a b i l i t i e s t h a t differences between e n z y m a t i c activities in the presence of m h l b l t o r s or gas mixtures a n d c o n t r o l s are significant
Chem.-Biol Interactions, 3 (1971) 383-396
389
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVES AND N-OXIDES T A B L E IV
EFFECT OF PRETREATING RATS ON THE LIVER MICROSOMAL CONVERSIONOF RETRORSINE TO PYRROLE DERIVATIVES AND TO N-OXIDES in Htro a Pretreatment
Enzyme acttvtty ( % o f mean control va?ues ± S E ) Pyrrole dertvattves
Phenobarbltone (3 x 100 mg/kg, 1 p ) D D T (1 × 75 mg/kg, 1 p ) 3-Methylcholanthrene (1 × 2 0 mg/kg, l p ) Amanoacetonltrlle (3 × 100 mg/kg, 1 p ) N T P (1 × 2000 mg/kg, p o ) Retrorslne (1 × 35 mg/kg, i p ) Sucrose diet (2 days) Protein-free diet (3 days) Starvatmn (2 days)
311 ± 6 ( P < 0 0 0 1 ) 407±6(P<0001)
Slgntficance c
N-Oxide
d a
95 ~: 7 ( N S ) a
232-k3 (P<0001) a 203 ± 3 ( P < 0 0 0 1 ) a
P<001 P<001
116± 6(NS) a
NS
122 Jz 6 b (P < 0 05) a 178 ± 2 b ( P < 0 0 0 1 ) d 63 ~ 5 b ( P < 0 0 1 )
a
5 0 ± 12b ( P < 0 0 5 ) a 39 ± 2 b (P < 0 001) a 69 ± 3b (P < 0 01) a
a Each result represents the mean of 4 experiments b Retrorsme concentration 0 33 m M Incubation stopped after 60 m m c Probablhtles that differences between rates of formation of pyrrole derivatives and N-oxides are significant a Probablhtles that differences between enzymatic activities in pretreated animals and controls are slgmficant N S , not significant, l p , lntraperltoneally, p o , per os
The effects of substrate concentration were examined to see if differences could be revealed between the enzymatic pathways leading to the formation of pyrrole derivatives and N-oxides The apparent Km values towards retrorsme for the formation of pyrrole derivatives and N-oxides were found to be very stmdar (Table V) Accurate estimations of the Km values with respect to monocrotaline and lasiocarpme N-oxides proved to be impracticable This was due m the case of monocrotahne to the low rate of enzymatic conversion to monocrotallne N-oxide and in the case of laslocarpme, to the low molar extinction coefficient given by its N-oxide, when estimated by the method at present available 1°. When the N-oxides of retrorsine, laslocarpine, monocrotaline or hehotrme, free from the parent alkaloids, were incubated with the mlcrosomal preparations, no conversion to pyrrohc derivatives could be detected The results of a smgle experiment to investigate the conversion of a number of different pyrrohzidme alkaloids to pyrrollc derivatives by a rat-hver microsomal prep° aranon are shown in Table VI. The noncyclic dlester lasiocarpine and the cyclic dlesters having saturated pyrrohzldlne ring structures, platyphylhne and rosmarmine, gave the highest yields of pyrroles, while semisynthetlc short-chain dlesters such as dlacetyl retronecine were amongst those alkaloids giving the lowest yield. Rat-lung microsomal preparations were unable to convert either monocrotahne or retrorsine to pyrrole derivatives Such lung preparations were able to N-demethylate N,N-dImethylanlhne The specific enzymatic activity for this reaction was about 30 % Chem-Btol Interactwns, 3 (1971) 383-396
390
A.R.
MATTOCKS, I. N. H WHITE
TABLE V APPARENT
Kra V A L U E S F O R T H E C O N V E R S I O N OF R E T R O R S I N E ~ M O N O C R O T A L I N E
AND
LASIOCARPINE TO
P Y R R O L E D E R I V A T I V E S A N D T H E C O N V E R S I O N OF R E T R O R S I N E T O N - O X I D E a
Substrate
Km (mM, mean 4. S E ) Pyrrole derivative N-Oxide
RetrorsIne Monocrotahne Laslocarpxne
0 30 4. 0 02 b 0 60 4, 0 06 0 18 4- 0 02
0 32 4. 0 06 b ---
a E a c h value represents the m e a n o f 4 experiments b N o significant d~fference for Km values towards the f o r m a t i o n o f pyrrole derivatives a n d N-oxides f r o m retrorslne
that of hepatic mlcrosomes. When examined by electron microscopy, the mlcrosomal fractions from both organs appeared to be similar. DISCUSSION
The rat-liver enzymes responsible for converting pyrrohzldlne alkaloids to pyrrole derivatives and to N-oxides have their highest specific activities an the mlcrosomal fraction They have many of the characteristics of mlcrosomal mlxed-functlon oxldases 16, which are concerned with the metabohsm of numerous foreign orgamc compounds Conversion of the alkaloids to pyrrole derivatives has previously been shown to take place in liver-tissue slices 4 as well as m the mlcrosomal fraction of rat hver 6'7. The act~wt~es of the mlcrosomal enzymes are influenced by a variety of factors such as sex, age, nutritional status or drug pretreatment of the ammal Iv-19 In the conT A B L E VI A C O M P A R I S O N OF T H E E X T E N T OF E N Z Y M A T I C C O N V E R S I O N O F A N U M B E R O F P Y R R O L I Z I D I N E
ALKALOIDS
T O P Y R R O L E D E R I V A T I V E S BY R A T - L I V E R M I C R O S O M E S In vitro
Alkaloid a
Relative enzymatic activities e (]ormatton of pyrrole derivatives as % of retrorsme)
Acute toxicity LD5o (mg/kg)
Laslocarplne Platyphylhne Rosmarlnlne
260 125 110
88 (ref 3, p 145) N o acute hepatotoxlclty (ref 4) N o acute hepatotoxlclty (ref 4)
Retrorsme Seneclonme Isoseneclomne Anacrotlne
100 100 74 66
38 (ref 3, p 145) 85 (ref 3, p 143)
Monocrotahne Senklrklne Hehotrlne Dmcetyl retroneclne
27 20 19 18
91-175 (ref 3, pp 142, 145) 200-300 (ref 40) 274 (ref 3, p 145) > 900 (ref 29)
Strlgosine b M o n o c r o t a h n e epox~de 7-B-Hydroxy-l-methylene pyrrohzldlne
15 0
N o acute hepatotoxlclty (ref 5)
0
N o acute hepatotoxlclty (ref 25)
a C o n c e n t r a t i o n o f alkaloids was 0 33 m M b C o n c e n t r a t i o n 0.66 m M ¢ Reaction stopped after 60 m l n
Chem-Btol Interactions, 3 (1971) 383-396
CONVERSIONOF ALKALOIDSTO PYRROLEDERIVATIVESAND N-OXIDES
391
version of the pyrrohzldme alkaloids to pyrrole derivatives and to N-oxides, the sex difference with respect to the formation of pyrrole derivatives is greater than that for N-oxides The conversion of retrorsme to pyrrole derivatives and to N-oxides requires oxygen and reduced NADP. N A D H appears to play no direct role in the reaction sequence but in common with many of the mlcrosomal mixed-functlon oxldases has a stablhsmg influence on the enzyme system 1° The usual cations which activate the liver mlcrosomal enzymes, e g. Mg 2+ and Ca z+ (ref 21), stimulate the production of pyrrohc metabohtes from retrorsme It IS of Interest that Mn 2+ potentiates enzymatic activity even in the presence of an optimal concentration of Mg 2+ Thls was found also to be true for Ca 2+ Since chemically reduced N A D P H was employed, the action of these ions cannot be on the N A D P H generating system Ca 2+ did not further activate the enzyme when Mn 2+ was also present, so both might be acting at similar sites in the mlcrosomal complex.
Inhlbltors A number of compounds which inhibit liver mlcrosomal drug-metabohsmg enzymes, such as SKF 525A and carbon monoxide, also inhibit the formation of pyrrohc and N-oxide metabohtes Inhibition of the former is significantly greater than that of the latter when carbon monoxide is used Mlcrosomal systems responsible for N-oxidation do not always behave in this way For example, the formation of N,N-dlmethylamhne-N-oxlde from N,N-dlmethylanlhne by pork-hver mlcrosomes is little affected by SKF 525A or carbon monoxide 2z Nicotlnamlde was at one time routinely included m incubation mixtures by many workers investigating mlcrosomal drug metabolism to inhibit pyridme nucleotadases 23. In our experiments, nlcotmamlde consistently inhibited the formation of pyrrole derivatives but had little effect on the production of N-oxldes The action of mcotmamide on the mlcrosomal enzymes is complex For some reactions It acts as a competitive or a mixed type of inhibitor 24, while for others it enhances enzymatic activity25.
Mlcrosomes from pretreated rats Hepatic mlcrosomes from rats pretreated with phenobarbitone converted retrorsine much more rapidly to pyrrole metabohtes than the corresponding control animals The formation of N-oxide was increased to a smaller extent These results were obtained whether phenobarbltone was administered by mtraperltoneal injection or given as a solution in the drinking water The action of DDT was similar to that of phenobarbitone, the formation of pyrrole derivatives being induced to a greater extent than the formation of N-oxides In contrast to D D T and phenobarbltone, the polycychc hydrocarbon 3-methylcholanthrene, which is also capable of inducing a number of hepatic drug-metabollslng enzymes 17, failed to significantly enhance the production of either pyrrohc or N-oxide metabohtes The amounts of pyrrohc metabolltes formed from retrorslne were decreased when mlcrosomes were prepared from rats which had been starved or fed a sucrose or protein-free diet. These findings are consistent with the results of other workers
Chem-Btol Interactions, 3 (1971) 383-396
392
A R MATTOCKS, I. N. H. WHITE
who have shown that protein-depletion of rats leads to a reductmn in the hver mlcrosomal drug-metabohsing enzymes Is. The acute toxicity of retrorslne in vtvo was also decreased in rats fed on a sucrose diet 26, a result which is compatible with the view that acute toxicity is related to the microsomal metabolism of this alkaloid Amlnoacetonitrlle has been reported as offering partial protection against the acute necrotic effect m livers of rats following administration ofdImethylnItrosamlne 27 The action of amlnoacetonltrde was investigated to see if it could reduce the formation of pyrrole derivatives and thereby possibly protect the animal against the acute hepatotoxtc effects of the pyrrohzldlne alkaloids. However, the latter seems unlikely since pretreatment with th~s compound slightly actwated the mlcrosomal system. N T P IS a mixture of aryl phosphates which inhibit esterase activity 28. Rats were pretreated with this material to assess the importance of liver esterases in the metabolism of the pyrrollzidlne alkaloids The result was a significant increase m the microsomal metabohsm of retrorsine to pyrrole derivatives The toxicity to rats of diacetyl retronecme which is very easily hydrolysed was increased after treatment with N T P 29. However, it is not certain whether these results were due to esterase inhibition or to mductmn of the mlcrosomal enzymes by some constituents of the N T P mixture. It has been proposed that the initial pyrrollc metabohtes from the hepatotoxlc alkaloids might act as blfunctional alkylatlng agents 4's. Since these could react with nucleophlhc groups In proteins, the mlcrosomal enzymes themselves might be attacked Pretreatment of rats with retrorslnejust below an LDso dose brought about a decrease in the subsequent production of pyrrole by the isolated mxcrosomes m vitro The significance of this result is difficult to assess, since some hepatic cell necrosis had probably occurred, but it is evident that 20 h after a near-lethal dose ofretrorslne the system retains a high capacity for producing pyrrohc metabohtes M e t a b o h s m o f other alkaloids
A number of pyrrohzldlne alkaloids are converted into pyrrole derivatives by rat-hver microsomal enzymes Two d~fferent types of pyrrohc metabohtes have been identified Alkaloids which are esters of unsaturated amino alcohols, including laslocarplne, hehotrlne, retrorslne, seneclonlne, anacrotme, monocrotahne, senkirklne, and the semlsynthetic compounds lsoseneclonlne and dlacetylretronecme, give dlhydropyrrohzlne esters which are more reactive than the parent alkaloids and are capable of acting as alkylating agents 4's'7. As prewously reported, the alkaloids platyphylhne 5 and rosmarmlne% which have saturated pyrrohzldlne rings, gave relatively large amounts of pyrrohc derivatives. We have evidence that in rosmarlnme and platyphylhne ring A is dehydrogenated giving pyrrole derivatives which are quite different from those from the hepatotomc alkaloids (Fig. 2) (ref. 26). Both the ester groups in I are highly labile due to conjugation with the nitrogen, whereas the ester II xs relatively unreactive Platyphylhne and rosmarinlne have not been shown to be hepatotoxlc. Platyphylhne does not produce acute liver damage in the rat 3°'31, and has been used chnlcally as a substitute for atropine without causing any long-term hepatic or renal damage 32. Chem-Btol Interactions, 3 (1971) 383-396
CONVERSIONOFALKALOIDSTO PYRROLEDERIVATIVESANDN-OXIDES I
OCO
I
~
393
I
OCO
(~ I COOH I-
CO0
R
l
CH2OCO
L
R
l
CH2OCO
(b)
Fig 2 Conversxonto pyrrohc metabohtes of (a) toxic pyrrohzldme alkaloxds (such as seneclonme) and (b) nontoxic alkaloids (such as rosmarmme)
All the alkaloids which are known to be hepatotoxlc form pyrrole derivatives of the first, reactive type W~thm this group, there is some relaUonshlp between amounts of pyrrohc metabohtes formed m vitro from the alkaloids and their acute hepatotoxlcit]es to the rat m vtvo, although the latter are taken from several sources and may not necessarily be accurate for our strata of rats Thus retrorsme, with high toxicity, gwes a large amount of pyrrolic metabohtes, whereas hehotrme, which has low toxicity, gives much less There are exceptions, and the correlations are better when hver metabohte levels are measured tn vtvo 26. Lasiocarpme has about half the acute tOXlClty of retrorslne when measured m vtvo (ref. 3, p. 145), whereas the amount of pyrrole formed m vttro is more than double that from retrorsme. Laslocarpme has also been shown to give consistently stronger pyrrole colour than retrorsme m rat-hver tissue shces 4 and in human-embryo hver-txssue shces 33 However, the amounts ofpyrrole m rat hver 2 h after an LDso dose of las10carpme or retrorsme are similar 26 It therefore seems hkely that some factor other than mlcrosomal enzymatic activity limits the rate of pyrrole formation from laslocarpme m vtvo. One possibility is that the noncychc dlester laslocarpme is more susceptible to attack by hver esterases m vtvo and this reaction competes with the production ofpyrrole denvattves Seneclomne, which differs from retrorsme by one hydroxyl group, gives as much pyrrole as retrorsine but is less toxtc. The effect of a change in the stereochemistry of the acid moiety is seen by comparing seneclonlne and lsoseneclonlne, which are esters of retronecine with (-r) and (--) seneclc acid, respectively 26 Stngosme is a saturated alkaloid 34 of which too little was available for further study. T o x t c z t y to l u n g s
Monocrotahne and fulvlne, when administered to rats m vtvo at acute LDso doses, cause lung lesions m addition to their hepatotoxxc actions 2,as. Retrorsme generally has no effect on lung tissue As previously reported 29, the conversion of e~ther monocrotahne or retrorsme to pyrrole derivatives could not be demonstrated using lung microsomal preparations. The inability of human-embryo lung-tissue slices Chem-Blol Interacttons,
3 (1971) 383-396
394
A R MATTOCKS, I N H. WHITE
to convert laslocarplne, fulvlne and retrorsme to pyrroles has also been reported 33. Synthetic pyrrohc esters can cause lung damage similar to that produced by monocrotahne and fulv]ne 29'36, and it is likely that some metabohtes formed from these alkaloids in the liver are sufficiently stable to escape Into the circulation and cause lung lesions Metabohc pathways The apparent Km values for formation of pyrrole derivatives and N-oxides in rat-hver mlcrosomal preparations are similar, but this does not indicate whether the two metabohtes are formed independently or if the N-oxide Is an intermediate in the formation of pyrrole derivatives Although rat-liver slices incubated m vitro produce small amounts of pyrrole derivatives from the N-oxides 4, this is not demonstrable using the isolated mlcrosomal enzyme system This finding is in agreement with the results of other workers 7 Values of Km towards retrorslne pyrrole and N-oxlde formation are 6-7 times higher than values obtamed with respect to the hydroxylatlon of small aromatic molecules, e g. aniline, but are of the same order as those for reactions involving more complex compounds, e g the N-demethylatlon of ethylmorphlne 37. Neither the pyrrollc or N-oxide pathways are greatly affected by pretreatment of rats with 3-methylcholanthrene Both are mduced by pretreatment with phenobarbltone or D D T , but the induction of pyrrole formation is greater than that of N-oxide Nlcotlnamlde has an inhibitory effect only on pyrrole formatton Carbon monoxide inhibits the formation of pyrrole derivatives more than that of N-oxides While not conclusive, the results favour the view, shared by other workers 7, that pyrrohc metabohtes are formed from the alkaloids through a separate enzymatic pathway and not through the N-oxides. Possible reaction mechamsms The formation of pyrrohc derivatives means that the pyrrohzldme ring is dehydrogenated The mechanism of the dehydrogenation brought about by rat-hver fractions is not known, but it may be pointed out that this need not be a new type H
5
3 H
I
CH3 II
III
IV
Fig 3 Hypothetical conversion o f u n s a t u r a t e d pyrrohzldlne alkaloids to dlhydropyrrollzlnes (IV), initiated by enzymatic hydroxylatlon (of I) or d e m e t h y l a t l o n (of II) F o r slrnpllClty, ring substltuents n o t directly involved m the reactions have been omitted
Chem -Btol Interactions, 3 (1971) 383-396
CONVERSION OF ALKALOIDS TO PYRROLE DERIVATIVESAND N-OXIDES
395
o f m l c r o s o m a l reaction R e a c ti o n s c o m m o n l y catalysed by m l c r o s o m a l enzymes, such as N - o x l d a t m n or, m o r e p r o b a b ly , C-hydroxylatton, could be the m l t l at l n g process E n z y m a t i c h y d r o x y l a t l o n at positions 3 or 8 o f the d e h y d r o p y r r o l l z l d l n e ring (F~g. 3, I) should lead to the d l h y d r o p y r r o l l z l n e structure (IV) by a s p o n t a n e o u s sequence o f chemical (nonenzymatlc) reactions. E n z y m a t i c N - d e m e t h y l a t l o n o f alkaloids h a v m g o t o n e c l n e - t y p e bases (II) should lead to (III) which again w o u l d s p o n t a n e o u s l y give (IV). Wh ereas the p y r r o l m d l n e N-oxides are readily d e h y d r a t e d by heat or chemical r e a c t m n s 6'38'39 to pyrroles hke IV (Fig. 3), this reaction is n o t b r o u g h t a b o u t by the m l c r o s o m a l e n z y m e system, and it appears likely that these c o m p o u n d s are 'detOXlCatmn p r o d u c t s ' which, being highly water soluble, are rapidly excreted. F o r m a t i o n o f the different pyrroles (II, Fig 2) f r o m r o s m a r l n l n e an d platyp h y l h n e p r o b a b l y depends u p o n loss o f the car b o x y l at e a m o n at C-7 as p a r t o f a r e a c t m n sequence initiated by enzymatic h y d r o x y l a t l o n in rlng A. ACKNOWLEDGEMENTS W e t h a n k D r D. H G CROUT (Exeter University) for specimens o f senklrkme -J
/
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