Arsenic Uptake and Metabolism by the Alga - ACS Publications

8 Arsenic Uptake and Metabolism by the Alga Tetraselmis Chui N. R. BOTTINO—Department of Biochemistry and Biophysics, Texas A & M University, College Station, TX 77843 E. R. COX—Department of Biology, Texas A & M University, College Station, TX 77843 Downloaded by CORNELL UNIV on May 11, 2017 | http://pubs.acs.org Publication Date: January 12, 1979 | doi: 10.1021/bk-1978-0082.ch008

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K. J. IRGOLIC, S. MAEDA , W. J. McSHANE, R. A. STOCKTON, and R. A. ZINGARO—Department of Chemistry, Texas A & M University, College Station, TX 77843 Not too many years ago there was no great public or even s c i e n t i f i c concern about substances present in very low concentrations in the a i r , water or soil. Knowledge about their presence was not at hand. Now, because of the efforts of many analytical chemists a number of these trace elements and their compounds have been identified and quantified. We know now that a whole spectrum of compounds i s present at trace level concentrations in the environment. We also know that many of them are, or may be potentially harmful. Arsenic i s one of the elements which is widely distributed in the earth's environment. Arsenic has a complicated chemistry. It forms a great variety of organic and inorganic compounds of variable potency with regard to their effects on l i v i n g organisms. Arsenic compounds are amenable to chemical transformation through interactions with biological systems. It has been known for almost one hundred years, that molds convert the arsenic present in green paint pigments into a volatile arsenic compound which was identified as trimethylarsine (1). The biological methylation of arsenic compounds producing methylarsines is now a well-established fact (2, 3, 4, 5). Methylcobalamin has been implicated as the methyl donor (2), but recently, Cullen and coworkers (5a) have made the Tmportant discovery that when L-methionine-methyl-d is added to certain microbial cultures, the CD label is incorporated into the evolved arsenic. This is good evidence that S-adenosylmethionine i s involved in the biological methylation process. Lunde (6) found arsenic compounds in the lipids of marine and limnetic organisms. Acid hydrolysis of the arsenic-containing l i p i d fractions produced water soluble, organic arsenic compounds. The isolation of arsenobetaine from rock lobsters was reported by Edmonds et al. (7) in 1977. These findings prove that transformations more complex than simple methylation do occur to 3

3

Present Address: Department of Applied Chemistry, Faculty of Engineering, Kagoshima University, Kagoshima 890, Japan. 1

0-8412-0461-6/78/47-082-116$05.00/0 © 1978 American Chemical Society Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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BOTTiNO E T A L .

Arsenic Uptake by Alga

Downloaded by CORNELL UNIV on May 11, 2017 | http://pubs.acs.org Publication Date: January 12, 1979 | doi: 10.1021/bk-1978-0082.ch008

a r s e n i c compounds i n b i o l o g i c a l

117

systems.

A r s e n i c Incorporation and Algal Growth. Three years ago an i n v e s t i g a t i o n on the metabolism o f arsenate by marine algae was i n i t i a t e d a t Texas A&M U n i v e r s i t y . The goal o f the project was and s t i l l i s the i s o l a t i o n , i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f organic a r s e n i c compounds formed by the algae from arsenate under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . Preliminary experiments with a v a r i e t y o f marine algae l e d to the use o f Tetraselmis ehui, a green f l a g e l l a t e (Chlorophyta), as a convenient experimental organism. This alga grows w e l l , takes up a r s e n i c e f f i c i e n t l y and i s r a t h e r i n s e n s i t i v e toward mechanical shock which accompanies the harvesting o p e r a t i o n s . T. ehui was found to t o l e r a t e well exposure to 10 ppm As(arsenate) when grown i n a r t i f i c i a l sea water, and i n f a c t , at the beginning o f the s t a t i o n a r y phase the growth medium contained more than one m i l l i o n c e l l s per ml (8). The a r s e n i c uptake by T. ehui depended on the l i g h t i n t e n s i t y . At a l i g h t i n t e n s i t y o f 12,500 lux the a r s e n i c content o f the c e l l s increased f i r s t to a pronounced maximum w i t h i n several days and then decreased r a p i d l y to a very low l e v e l . At 7000 lux the a r s e n i c l e v e l i n the c e l l s remained at h a l f the concentration reached at 12,500 l u x . The d e t a i l e d causes f o r t h i s behavior remain unknown, but i t can be p o s t u l a t e d that s i n c e arsenate absorption i s an endergonic process (9) i t might compete with c e l l growth f o r the a v a i l a b l e photosynthetic energy. Large-Scale Algal Growth and A r s e n i c - P r o t e i n I n t e r a c t i o n s . The c h a r a c t e r i z a t i o n o f the a r s e n i c - c o n t a i n i n g compounds present i n τ. ehui r e q u i r e d the i s o l a t i o n o f 300-500 mg amounts o f these compounds f o r f u r t h e r p u r i f i c a t i o n and a n a l y s i s . This could not be done with the t e s t - t u b e c u l t u r e s used i n the preceding ex periments. Consequently, an alga growing f a c i l i t y was con s t r u c t e d t o r a i s e T. ehui under c o n t r o l l e d c o n d i t i o n s i n c l u d i n g p r o t e c t i o n from other organisms such as b a c t e r i a . The f a c i l i t y c o n s i s t s o f a converted c o l d storage room, which houses four 1 5 0 0 - l i t e r tanks. This c u l t u r e room i s a i r - c o n d i t i o n e d , well i n s u l a t e d and i s kept under p o s i t i v e a i r pressure. Ultraviolet l i g h t s are s t r a t e g i c a l l y placed to minimize b a c t e r i a l contamin ation. The tanks are i l l u m i n a t e d by banks o f f l u o r e s c e n t l i g h t s . B a l l a s t s are mounted outside the c u l t u r e room t o reduce the heat load on the a i r c o n d i t i o n e r . The growth medium i s mixed i n a c o n t r o l room. The tanks can be f i l l e d , i n o c u l a t e d , aerated, sampled and harvested without e n t e r i n g the c u l t u r e room, through an appropriate system o f pipes and v a l v e s . Each large tank produces approximately one l i t e r o f t i g h t l y packed algae w i t h i n 20 days. Figure 1 i l l u s t r a t e s the t y p i c a l type o f experiments run with the l a r g e - s c a l e growing f a c i l i t y . When A s - l a b e l e d d i s sodium hydrogen arsenate was added to the growth medium at the same time as the inoculum, the algae reproduced r a p i d l y f o l l o w i n g 7 4

Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

A N D ORGANOMETALLOIDS


ORGANOMETALS

lure 1. Large-scale growth and arsenic uptake by T. ehui instant ocean medium containing 10 ppm As (arsenate) added at the time of inoculation


8. ΒΟΤτίΝΟ E T A L .


an i n i t i a l l a g p e r i o d , i n a r t i f i c i a l sea water c o n t a i n i n g 10 ppm arsenate. In separate experiments, the arsenate was added to the growing c u l t u r e s during t h e i r exponential phase o r s t a t i o n a r y phase. In the experiment depicted i n Figure 1 the arsenate was added at zero time, i . e . , at the time the medium was i n o c u l a t e d . The curves show p e c u l i a r , but recurrent f l u c t u a t i o n s r e s u l t i n g from changes i n the rate o f i n c o r p o r a t i o n and e f f l u x o f a r s e n i c i n t o and out o f the c e l l s . The two phenomena seem to reach an e q u i l i b r i u m a t the time the a l g a l c e l l s reach the s t a t i o n a r y phase o f growth (about day 14). Other experiments (not shown) i n d i c a t e d that intake and e f f l u x o f ^ A s e q u i l i b r a t e d at approx imately the same l e v e l shown i n Figure 1, whether the ^ a r s e n a t e was added a t the time o f i n o c u l a t i o n , during the exponential growth phase, or during the s t a t i o n a r y phase. The a l g a l c e l l s were a l s o grown i n the presence o f arsenate, then c o l l e c t e d at a time when there were at l e a s t one m i l l i o n c e l l s per ml and the c e l l population d i d not change a p p r e c i a b l y for two or three days. The c e l l s were then c e n t r i f u g e d and washed repeatedly with an A s - f r e e medium to remove any arsenate adhering to the c e l l s u r f a c e s . The a l g a l c e l l s were then homogenized i n chloroform/methanol (2:1 v/v) and e x t r a c t e d repeatedly with t h i s solvent mixture u n t i l a l l the green pigments had been removed and the residue assumed a grayish-white appearance. Approximately one h a l f o f the a r s e n i c was i n the organic e x t r a c t ; the other h a l f remained i n the r e s i d u e . The a r s e n i c d i s t r i b u t i o n between these two phases v a r i e d somewhat from batch to batch. When the residue was t r e a t e d with 1 M HC1 and the r e s u l t i n g s o l u t i o n was d i s t i l l e d , a l l the a r s e n i c remained i n the d i s t i l l a t e as a r s e n i t e as i n d i c a t e d by polarography and by reduction to a r s i n e . Hot.water e x t r a c t e d considerable amounts o f a r s e n i c from the r e s i d u e . When four ml o f methanol were added t o one ml o f aqueous e x t r a c t the p r e c i p i t a t e contained a l l the a r s e n i c . When i n c r e a s i n g amounts o f Na2HAsÛ4 were added t o f i x e d volumes o f the water e x t r a c t and the r e s u l t i n g s o l u t i o n s were mixed with methanol, a l l the arsenate p r e c i p i t a t e d q u a n t i t a t i v e l y when 500 yg o f As or l e s s were added. A r s e n i c added i n excess o f 1 mg remained i n the supernatant. The a r s e n i c content of the p r e c i p i t a t e reached a maximum value o f approximately 1 mg As (Figure 2 ) , i n the p r e c i p i t a t e obtained from one ml of the aqueous e x t r a c t . Most o f the a l g a l growth experiments were c a r r i e d out with arsenate tagged with the gamma-emitting A s isotope. When the p r e c i p i t a t i o n s with methanol a f t e r a d d i t i o n o f n o n - r a d i o a c t i v e Na£HAs04 were repeated, but only the ' ^ A s - c o n t e n t o f the p r e c i p i t a t e s and the supernatants determined, the r e s u l t s summarized i n Figure 3 were obtained. Exchange o f n o n - r a d i o a c t i v e arsenate f o r the r a d i o a c t i v e a r s e n i c compounds occurred only a f t e r approximately 500 yg As(arsenate) had been added t o one ml o f aqueous e x t r a c t . Both the p r e c i p i t a t e formed upon methanol a d d i t i o n , and the supernatant gave p o s i t i v e ninhydrin and b i u r e t t e s t s i n d i c a t i n g the presence o f 7


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7 4


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ORGANOMETALLOIDS


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Figure 2. The arsenic content of the precipitates and supernatants obtained upon addition of arsenate and then methanol to the aqueous arsenic-containing extracts of the residue from the CHCls/CH OH extraction of T. ehui s



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BOTTiNO E T A L .


121

Figure S. The distribution of As activity between the precipitates and supernatants obtained upon addition of arsenate and then methanol to the aqueous arsenic-containing extracts of the residue from the CHCl /CH OH extraction of T. ehui 74

s

s


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amino groups and p r o t e i n . These observations are c o n s i s t e n t with the working hypothesis that arsenate forms a complex with a p r o t e i n located i n the algal c e l l membrane p r i o r to i t s chemical transformation by the biochemical apparatus o f the c e l l . The separation o f t h i s complex by chromatographic techniques i s i n progress. I s o l a t i o n and C h a r a c t e r i z a t i o n o f A s - C o n t a i n i n g L i p i d s . The combined organic s o l u t i o n s obtained by homogenizing the a l g a l c e l l s i n chloroform/methanol were dark green. The removal o f these pigments from the a r s e n i c compounds was d i f f i c u l t . The separation scheme which l e d t o the i s o l a t i o n o f an a r s e n i c - c o n t a i n i n g l i p i d f r a c t i o n free o f green compounds i s summarized i n Figure 4. A d d i t i o n o f water t o the e x t r a c t produced a chloroform l a y e r which contained a l l o f the a r s e n i c . Several p r e c i p i t a t i o n s with acetone removed the phospholipids and a l l the a r s e n i c from the chloroform phase (10). Gel f i l t r a t i o n chromatography on Sephadex LH-20 o f the green p r e c i p i t a t e produced green, a r s e n i c c o n t a i n i n g bands, which were then chromatographed on DEAE C e l l ulose employing a sequence o f solvents ranging from chloroform, chloroform/methanol, (9:1 v/v) chloroform/methanol/acetic a c i d (3:1:1 v/v/v) to a c e t i c a c i d . The brownish-green a r s e n i c f r a c t i o n s were f u r t h e r p u r i f i e d by preparative high pressure l i q u i d chromatography on S i l i c a gel with chloroform/methanol/ a c e t i c acid/water (17:3.5:2:1 v/v/v/v). A s l i g h t l y brown o i l was obtained. A n a l y t i c a l HPLC o f t h i s o i l on S i l i c a gel using a Hitachi Zeeman Graphite Furnace Atomic Absorption Spectrometer as an a r s e n i c - s p e c i f i c detector showed that a t l e a s t two a r s e n i c compounds were present (Figure 5). T h i n - l a y e r chromatography on S i l i c a gel H using a mixture o f chloroform methanol a c e t i c a c i d water ( 5 0 : 2 5 : 8 : 4 , v/v) (11_) as the developing solvent showed two major a r s e n i c - c o n t a i n i n g com pounds with Rf values o f 0.41 (compound A) and 0.61 (compound B) and one major component without a r s e n i c (compound C) with an Rf o f 0.85. F r a c t i o n A was the l a r g e s t , followed by f r a c t i o n s Β and C. Compound A co-chromatographed with phosphatidyl c h o l i n e , compound Β with phosphatidyl s e r i n e and compound C with phospha t i d y l ethanolamine and monogalactosyl d i g l y c e r i d e . A standard o f sulfoquinovosyl d i g l y c e r i d e was not a v a i l a b l e at the time these experiments were r u n , but i t s Rf i n the solvent system used would have been considerably lower than 0.41. Further experiments are i n process which involve the use o f other solvent systems, appropriate standards and c o l o r reactions f o r the r e c o g n i t i o n o f s p e c i f i c chemical groups. The f a t t y a c i d compositions o f compounds A, Β and C were determined by g a s - l i q u i d chromatography and are shown i n Table I. The most remarkable c h a r a c t e r i s t i c o f these data i s the extremely low l e v e l o f C20 to C22 h i g h l y unsaturated f a t t y a c i d s . Such acids have been found at much higher concentrations i n the phospholipids o f several algae (12). It i s p o s s i b l e that the


BOTTiNO E T A L .


TETRASELMIS CHUI centrifugation

l ! =

ι Growth Medium

CELLS


homogeniz at i o n C/M 2:1

E = = ^

J Residue

Water/Methanol

Filtrate

g e l - f i l t r a t i o n LH-20 C/M 3:1

I Discard

e

» As-CONTAINING BANDS

DEAE C e l l u l o s e Ion exchange C; C/M; C/M/A; A

1»

C M A W

= = = •

Chloroform Methanol Acetic Acid Water

Figure 4.

As-CONTAINING FRACTIONS s i l i c a gel C/M/A/W prep. HPLC H Discard

discard

ARSENIC COMPOUNDS

Scheme for the chromatographic isolation of an arsenic-containing phospholipid fraction



ORGANOMETALS


Figure 5. Chromatographic separation and detection of two arsenic compounds in a phospholipid fraction isolation from T. ehui. (HPLC Microporosil (25 cm); CHCl /CH OH/CH COOH/H O, 17:3.5:2:1;flowrate, 0.5 mL min^/l-min fractions; Hitachi-Zeeman AA Model 170-70,193.6 nm.) s

s

s

t


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ΒοττίΝΟ E T A L .

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Table I . Major F a t t y Acids o f P o l a r L i p i d s from Tetraselmis ehui

Weigh Percent TLC f r a c t i o n s y


Fatty Acid

Unfractionated

d/

A

Β

d/ C

4.3

5.2

d/

14:0

3.6

5.5

16:0

20.4

27.8

15.7

8.3

18:0

3.8

18.3

10.5

7.4

24:0

2.6

1.4

0.9

28.5

10.4

11.3

29.6

13:1

12.8

e/ 16:1 w7 e/ 18:1 w9

17.5

20:1 w9

10.2

18:2 20:4

1.7 11.9 2.8

3.9

5.7

0.4

3.1

17.4

5.4

8.3 21.2 2.0

17.0 1.8

1.2 3.3 0.1

2.3 1.8

f/ UNKNOWN

— .Only f a t t y a c i d s present a t a l e v e l o f 2% o r more are i n c l u d e d . — Chain l e n g t h : number o f double bonds, w = p o s i t i o n o f f i r s t i double bond counting from the methyl end. — TLC = t h i n - l a y e r chromatography w i t h a mixture o f chloroform: methanol:acetic acid:water (50:25:8:4, v/v) as developing solvent. ~^R o f Fractions*. A = 0.41; Β = 0.61; C = 0.85. T / l t may c o n t a i n other isomers. — R e t e n t i o n time r e l a t i v e t o 18:1 = 0.60, on s i l i c o n i z e d d i e t h y l e n e g l y c o l s u c c i n a t e p o l y e s t e r a t 170°C. f



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prolonged e x t r a c t i o n procedure might have caused t h e i r l o s s . Elemental analyses o f the a r s e n i c - c o n t a i n i n g l i p i d s a f t e r chromatography on S i l i c a gel (Figure 4) showed a r s e n i c present at a l e v e l o f 0.5 percent and confirmed the presence of C, Η, Ρ and Ν i n r a t i o s c h a r a c t e r i s t i c o f those in phosphatidyl c h o l i n e . Pure phosphatidyl arsenocholine would have an a r s e n i c content o f 8.8 percent. HPLC separation and enzymatic and chemical h y d r o l y s i s experiments are in progress to i s o l a t e the a r s e n i c - c o n t a i n i n g components from the a l g a l l i p i d f r a c t i o n . In 1976 at the I n t e r n a t i o n a l Conference on Environmental Arsenic at Fort Lauderdale, we suggested (13) that a r s e n i c could replace nitrogen i n c h o l i n e . The arsenocholine I could then become part of a l i p i d 2 bonded to the phosphate group of a phosphatidyl r e s i d u e .

CH.

CH.

CH -As-CH C00 3 i c

CH -As^CH CH -0H o

2

o

2

o

CH.

CH.

CH -0-C-R 2

i

I

CH-O-C-R CH -0-f-0-CH CH -As(CHj) 2

2

2

3

0

The r e s u l t s obtained thus f a r with the a l g a l l i p i d s are c o n s i s t e n t with t h i s hypothesis. The i s o l a t i o n by Edmonds e t a l _ . , o f arsenobetaine 3 from rock l o b s t e r s (7) shows that an a r s e n i c



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BOTTINO E T A L .


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compound very s i m i l a r to arsenocholine e x i s t s i n nature. I t has been observed (14, 15) that a r s e n i c - c o n t a i n i n g compounds from various organisms would y i e l d v o l a t i l e a r s i n e s upon reduction with sodium borohydride, but only a f t e r the sample had been d i gested with 2H sodium hydroxide. We subjected s y n t h e t i c samples o f arsenobetaine and arsenocholine f i r s t to sodium hydroxide d i g e s t i o n and then to sodium borohydride reduction t o determine i f methylarsines were generated. Arsenocholine, under these c o n d i t i o n s , was found to produce, at b e s t , only t i n y traces o f dimethylarsine or t r i m e t h y l a r s i n e , but arsenobetaine d i d r e a d i l y . The a r s e n i c - c o n t a i n i n g l i p i d s i s o l a t e d from T. ehui do form dimethylarsine or t r i m e t h y l a r s i n e under these c o n d i t i o n s . Be cause betaine i s not a common c o n s t i t u e n t o f l i p i d s , i t i s un l i k e l y that a r s e n i c i s incorporated i n t o l i p i d s i n the form o f arsenobetaine. Arsenocholine might, however, be capable o f being converted t o arsenobetaine. The question as to whether such a conversion i s s i g n i f i c a n t i n organisms, can be answered a f t e r the a r s e n i c - c o n t a i n i n g l i p i d s have been i d e n t i f i e d . The chemical s t r u c t u r e o f the a r s e n i c compounds formed by T. ehui from arsenate should soon be known. The s t a r t i n g mat e r i a l and the end products o f t h i s b i o l o g i c a l conversion w i l l then have been i d e n t i f i e d . Future work w i l l have to be c a r r i e d out to e l u c i d a t e the biochemical pathway o f t h i s transformation. I n v e s t i g a t i o n s o f t h i s nature w i l l f i n a l l y lead to the develop ment o f metabolic charts f o r a r s e n i c and other t r a c e elements s i m i l a r to those now a v a i l a b l e f o r l i p i d s , carbohydrates, n u c l e i c acids and p r o t e i n s . U n t i l such t r a c e element metabolic pathways have been worked o u t , the d e t a i l s o f the i n t e r a c t i o n s o f t r a c e elements with l i v i n g organisms cannot be understood. Basic r e search that would e v e n t u a l l y achieve t h i s important goal has been i n i t i a t e d i n several laboratories. Acknowledgement: These i n v e s t i g a t i o n s were supported by the National I n s t i t u t e o f Environmental Health Sciences (Grant No. 5 ROI ES01125), by the Robert A. Welch Foundation o f Houston, Texas, and by the Texas A g r i c u l t u r a l Experiment S t a t i o n . Literature Cited 1. 2. 3.

4. 5.

Challenger, F . , Chem. R e v . , (1945) 36, 315. McBride, B. C., and Wolfe, R. S., Biochemistry, (1971) 10, 4312. Schrauzer, G. N . , Seek, J. Α . , H o l l a n d , R. J., Beckham, T . Μ., Rubin, Ε. Μ., and S i b e r t , J. W., B i o i n o r q . Chem., (1972) 2, 93. R i d l e y , W. P . , D i z i k e s , L., Chek, Α . , and Wood, J. Μ., Environ. Health P e r s p e c t . , (1977) 19, 43. Wood, J. Μ., S c i e n c e , (T974) 183, 1049.



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ORGANOMETALS

AND ORGANOMETALLOIDS

5a. C u l l e n , W. R., Froese, C. L . , L u i , Α., McBride, B. C., Patmore, D. J., and Reimer, Μ., J. Organometal. Chem., (1977) 139, 61. 6. Lunde, G . , E n v i r o n . Health P e r s p e c t . , (1977) 19, 47. 7. Edmonds, J. S., F r a n c e s c o n i , Κ. Α . , Cannon, J. R., Raston, C. L . , S k e l t o n , B. W., and White, Α. Η., Tetrahedron L e t t . , (1977) (18) 1543. 8. B o t t i n o , N. R., Newman, R. D . , Cox, E . R., S t o c k t o n , R., Hoban, Μ., Zingaro, R. A. and Irgolic, K. J., J. Exp. Mar. Biol. Ecol., in p r e s s . 9. B l a s c o , F., P h y s i o l . V e g . , 13 (1975) 185. 10. Kates, Μ., "Techniques o f L i p i d o l o g y : I s o l a t i o n , Analysis and I d e n t i f i c a t i o n o f L i p i d s " . American E l s e v i e r P u b l i s h i n g Co., New York, Ν. Υ . , 1972; p. 393. 11. S k i p s k i , V. P . , Peterson, R. F., and B a r c l a y , Μ., Biochem. J., (1964) 90, 374. 12. N i c h o l s , B. W., "Comparative L i p i d Biochemistry o f Photo s y n t h e t i c Organisms", i n Harborne, J. B . , e d . , "Phyto chemical Phylogeny". Academic P r e s s , London, 1970, p. 105. 13. I r g o l i c , K. J., Woolson, Ε. Α . , S t o c k t o n , R. Α . , Newman, R. D . , B o t t i n o , N. R., Zingaro, R. Α . , Kearney, P. C., P y l e s , R. Α . , Maeda, S . , McShane, W. J. and Cox, E. R., Environ. Health P e r s p e c t . , (1977) 19, 61. 14. C r e c e l i u s , Ε. Α . , Environ. Health P e r s p e c t . , (1977) 19, 147. 15. Edmonds, J. S . , and F r a n c e s c o n i , Κ. Α . , Nature, (1977) 265, 436.

Discussion Y. K. CHAU (Canada Centre f o r Inland Water Research): How do you separate the methylation due t o b a c t e r i a which generate the n e t h y l a r s e n i c compounds from c o n c e n t r a t i o n o f a r s e n i c a l s by algae? IRGOLIC: F i r s t of a l l , we t r y t o keep the b a c t e r i a out of the algae. We checked whether any t r i m e t h y l a r s i n e i s formed i n our a l g a l c u l t u r e s . We couldn't f i n d any w i t h As-74 t r a c e r s . These c u l t u r e s a r e not completely b a c t e r i a f r e e , but we b e l i e v e most of the transformation i s done w i t h i n the a l g a l c e l l and not by b a c t e r i a . M. 0. ANDREAE (Scripps I n s t i t u t e o f Oceanography): A word o f c o n f i r m a t i o n . We t r i e d t o see i f there i s a c o n t r i b u t i o n by bac t e r i a by comparing c u l t u r e s and monocultures of the same organism, rhere was no measurable d i f f e r e n c e , so I t h i n k that b a c t e r i a do aot c o n t r i b u t e s i g n i f i c a n t l y i n the k i n d of systems that you use. Did you make any attempt t o i d e n t i f y t h e substance that was released by your a l g a l system a f t e r 5 days o r whenever that b i g peak occurred? IRGOLIC: No, but there i s one experiment which I d i d n ' t de s c r i b e . We took these algae, grew them i n 10 p a r t s per m i l l i o n arsenate, and then determined t h e i r a r s e n i c l e v e l . We then r e suspended the algae i n arsenate-free medium and found that between 50% and 75% of the a r s e n i c comes r i g h t out. We would l i k e t o know


8.

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what form the a r s e n i c i s ; i t could simply be arsenate. The a r s e nate has t o go through the c e l l w a l l , perhaps t o form an arsenate complex there. The c e l l does the t r a n s f o r m a t i o n ; i t i n c o r p o r a t e s the a r s e n i c perhaps i n t o the l i p i d s . I f you d i s t u r b the c e l l , the arsenate can migrate r i g h t back out. We i n t e n d t o do t h i s ; we have now the s e n s i t i v i t y t o determinate t h i s arsenate by p o l a r ography.


ANDREAE: Did you t r y t o deacylate the f r a c t i o n i n your l i p i d s o l u b l e e x t r a c t and see i f i t could be i d e n t i f i e d by e l e c t r o p h o r e s i s o r chromatography? IRGOLIC: We t r i e d some phospholipases w i t h the a r s e n i c act i v i t i e s p a r t i t i o n e d between the organic phase and the aqueous phase. We have not yet i n t e r p r e t e d the r e s u l t s . ANDREAE: We t r i e d some d e a c y l a t i o n o f a l g a l l i p i d s , and used the water e x t r a c t . C o e l e c t r o p h o r e s i s w i t h arsenocholine o r a r senobetaine was u n s u c c e s s f u l . G. E. PARRIS (Food and Drug A d m i n i s t r a t i o n ) : I s there any comment o r suggestion regarding formation o f a bond between a r s e n i c and the two-carbon u n i t i n b e t a i n e o r arsenobetaine o r a r senocholine? How does i t occur? IRGOLIC: Somehow we have t o go from arsenate t o whatever that organic compound i s . I f i t ' s arsenocholine o r arsenobetaine, t h a t i s s i m i l a r . But i f there are many steps i n between, we r e a l l y can't t a l k i n t e l l i g e n t l y about what i s happening unless we understand that metabolic pathway. There i s some i n d i c a t i o n t h a t organoarsenic compounds produced by transformations i n marine o r ganisms, e.g., shrimp o r c r a b s , do not seem t o be t o x i c when i n gested by man, as shown by Dr. C r e c e l i u s ["Methods and Standards f o r Environmental Measurement", W. H. K i r c h h o f f , ed., Proc. E i g h t h M a t e r i a l s Res. Symp., NBS Spec. P u b l . 464, Washington, D.C., 1977, p. 495]. W. R. CULLEN ( U n i v e r s i t y o f B r i t i s h Columbia): A compound i s o l a t e d from A t l a n t i c f i s h by Environment Canada was repeatedly p u r i f i e d , and we got back an a n a l y s i s r e c e n t l y ; i t had no a r s e n i c i n i t . B a s i c a l l y , i t was b e t a i n e o r something very l i k e b e t a i n e . We suggest that somehow there i s probably an arsenate that gets a s s o c i a t e d w i t h the b e t a i n e i n some way and can be l o s t through purification. IRGOLIC: I t was i n t e r e s t i n g t o hear Dr. McBride mention the a s s o c i a t i o n which then breaks up. We might see something l i k e t h i s i n some o f the c e l l w a l l s ( e x t r a c t r e s i d u e s ) . U n f o r t u n a t e l y , I t h i n k we are i n the dark about these a s s o c i a t i o n s . RECEIVED

August 2 2 ,

1978.


Arsenic Uptake and Metabolism by the Alga - ACS Publications

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