Factors controlling the inhibitory effects of 3,4-benzo(a)pyrene on the chlorococcal alga Scenedesmus quadricauda

Arch. Hydrobiol. Suppl. 73,2(A\gologicaI Studies 43) 281-296 Stuttgart, September 1986

Factors controlling the inhibitory effectsof 3,4-benzo(a)pyrene on the chlorococcal alga

Scenedesmus quadricauda

By VILÉM ZACHLEDER,

Institute of Microbiology ÈSAV, Dept. of Autotrophic Microorganisms, Tøeboò,Czechoslovakia,

ECKARD WITfENBURG and SIBYLLE ABARZUA

Wilhelm-Pieck-University Rostock, Sect. of Biology, Rostock,German Democratic Republic

With 8 figures in the text

Abstract: The extent of benzo(a)pyrene (BP) inhibitory effects on macromolecular synthesesand cell-cycle characteristics was studied in synchronous populations of the chlorococcal algaScenedesmus quadricauda grown under various light conditions. The inhibitory effects of BPdecreases with increasing density of the cen culture and with a decrease in the incidentÍrradiance of cultures; the inhibition was completely prevented by darkening of the cens.Mean irradiance per cen was decisive for the extent of BP inhibitory effects irrespective ofthe combination of incident Írradiance and culture density. A certain time period of algal cengrowth in light was required for BP to become inhibitory active. The extent of inhibitoryeffects, therefore, tended to increase with the time of BP presence during the light period.BP added to the algal suspension was adsorbed quantitatively and rapidly by the cen surfaceand the extent of BP effects was related, under the given mean Írradiance and the length oflight interval, tó the BP concentration per cen rather than to BP concentration per volume ofnutrient medium.

Key words: 3,4-Benzo(a)pyrene - cen concentration control - inhibitory effects - meanirradiance control - Scenedesmus quadrjcauda.

PDC*: SS 122; BN 01, 02, 03; PY 06; BC 061, 081, 082, 102; CH 114; PH 08; GC 02,30; ME 01, 08, 10; UC 01, 03.

Abbreviations: BP = 3,4-benzo(a)pyrene, PhAR = photosynthetically active radiation.

Introductiou

The consequences of treatment of the chlorococcal a1ga Scenedesmus quadricaudawith 3,4-benzo(a)pyrene (BP) were described previously (ZACHLEDER et a1. 1983).The transcription activity of the genome was found to be inhibited and, conse-

* Phycological Documentation Code - see: AIgological Studies 9: 450-481, 1973.

0342-1120/86/0073-0281 $ 4.-@ 1986 E. Schweizerbart'scbe Verla.sbuchhandlun.. D-7000 Stutt.ar! 1

282 VILÉM ZACHLEDER, ECKARD WITfENBURG and SIBYLLE ABARZUA

quently, the inhibition of both RNA and protein syntheses occurred. The cellularprocesses, e.g., DNA replications, mitoses, protoplast fissions, and their induc-tions were affected under certain conditions as well. In the above mentioned paper(ZACHLEDER et al. 1983), gradation of inhibitory effects was attained by increaseof concentrations of BP per ml applied to the cultures grown under constantgrowth conditions and cell mass was kept constant during the experiments.However, during preparation of these experiments, it was found that the extent ofBP inhibitory effects varied markedly according to the culture conditions used.Therefore, in the case of BP, other factors rather than mele molar concentrationseem to play a role in determining its inhibitory activity.

The aim of this paper is to define these factors and find how they aremanifested in interactions between BP and algal cells.

We used synchronous populations of Scenedesmus quadricauda grown atvarious culture densities and/or various levels of incident irradian~e. Furthermore,modification of the inhibitory effects of BP by the duration of its presence in cellswas examined. The courses of DNA, RNA, protein and chlorophyll accumulMionas well as the inductions of nuclear and cellular divisions were measured to defineBP inhibitory effects.

Material aod methods

Organism

The chlorococcal alga Scenedesmus quadricauda (TuRP.) BRÉB. strain GREIFS-WALD/15 was obtained from the culture collection kept at the lnstitute of Experi-menta! Botany, Prague, Czechoslovakia (now at the lnstitute of Botany, Tøeboò,Czechoslovakia) .

Culture conditions

The cultures were synchronized by altemating light and dark intervals (14:10h).The suspensions of synchronous cells of Scenedesmus quadricauda were cultivatedin plate-parallel vessels illuminated from one side. Batch cultures of asynchronousScenedesmus quadricauda were used for the determination adsorption velocity ofBP, its stability and distribution between the cells and the solution. Cultureconditions in this case corresponded to those published by ABARZUA et al. (1983).In some experiments glass cylinders of 400 ml volume were used. Incandescentlamps (500 W) served as the light source. Irradiance was 140-200 W. m-2 PhAR atthe surface of the cultivation cuvettes. Carbon dioxide concentration in theaerating gas mixture was maintained between 1.5 and 3% (v/v). The inorganicnutrient solution was that described by ZACHLEDER & ŠETLfK (1982). The culturecuvettes were submersed in a water bath at the constant temperature of 30°C.Batch cultures, semicontinuously or continuously diluted cultures were used for

Factors controlling the inhibitory effects 283

experiments. Details of culture equipment and conditions were the same as thosedescribed by DOUCHA (1979). Continuous illumination was employed duringexperiments with BP.

Determination of induction curves

The samples were withdrawn at one- or two-hour intervals during the growth of

synchronous cultures and aerated in the dark at 30°C. The samples were fixed byiodine kalium-iodide solution at the end of the control cell cycle and the respectivepercentages of the four-celled and eight-celled daughter coenobia and of undi-vided mother cells were estimated. (Note: Two-celled daughter coenobia are notformed under the given conditions.) The values were plotted against the time atwhich the samples had been darkened. The curves obtained are termed theinduction curves. The significance of the term "induction" for various. reproduc-tion processes was explained and the method for its determination was describedby ŠETLfK et al. (1972).

Cen number

The number o• cells, or the fractions o• induced quadruplet and octuplet daughtercoenobia, and undivided mother cells were counted in the Biirker countingchamber (produced by Meopta, Czechoslovakia).

Chemicals

AII chemicals used for the anaIyses were of anaIyticaI grade. The DNA, RNA,casein, and aIbumine for calibration assays were obtained from Serva HeideIbergLtd, FRG 3,4-bt?nzo(a)pyrene from FERAK Ltd. West BerIin. The other chemi-cals were supplied by Lachema Ltd., Prague, CzechosIovakia.

Total nucleic acid assay

The procedure of WANKA (WANKA 1962) as modified by LuKAvsKÝ et al. (1973)was employed in the assay of total nucleic acids. The samples were centrifuged in10 ml centrifuge tubes, which also served for storage of the samples. The sedi-ments of the a!gal cells sampled were stored under 1 ml of ethyl alcohol at -20°C.The algae were extracted 5 times with 0.2 N perchloric acid in 50% ethyl alcoholfor 50 min at 20°C and extracted three times with an ethanol-ether mixture (3:1) at70°C for 10 min. Such pre-extracted samples CaD be stored in ethyl a!cohol. Totalnucleic acids were extracted and hydrolysed by 0.5 N perchloric acid at 60°C for5 h. After hydrolysis, concentrated perchloric acid was added to reach the fina!1 N

concentration of perchloric acid in each sample. Absorbance of tota! nucleic acidsin the supematant was read off at 260 Dm.

VILÉM ZACHLEDER, ECKARD WITTENBURG and SIBYLLE ABARZUA284

DNA assay

The light-activated reaction of diphenylamine with hydrolyzed DNA, as describedby DECALLONNE & WEYNS (1976), was utilized. Diphenylamine reagents (4%diphenylamine in glacial acetic acid, w/v) mixed with the samples of total nucleicacid extract at the ratio of 1:1, were illuminated from two sides with fluorescentlamps (Tesla 40 W). The incident radiation was 20 W. m-2 from either side. After6 h of illumination at 40°C, the differences were identified between the absorbance

at 600 nm and that at 700 Dm.

RNA content calculation

The RNA content was ca1culated as the difference between tbe total nucleic acidcontent and the DNA content. Optimization of conditions for, assay of nucleicacids in algal cells was described in detail by ZACHLEDER (1984).

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Protein assay

The sediment remaining after nucleic acid extraction was used for protein contentdetermination. LOWRY'S Folin-phenol method was employed in protein estimation

(LOWRY et al. 1951).

ChlorophyU assay

The amounts o• chlorophylls a and b present were assessed in acetone extracts o•disintegrated algae. The calculation o• the chlorophyll content from absorbancesmeasured at 645, 664 and 720 nm was carried aut by the method o• McKINNEY(1941). The procedure, modified for Scenedesmus quadricauda, has beendescribed by LuKAvsKÝ et al. (1979).

~easureDment of kra~ance

In order to define the light conditions in suspensions of varying density andreceiving different incident irradiance, the mean irradiance (I) was determinedfrom the irradiance measured at the surface of the culture vessel (IJ and fromirradiance transmitted through the suspension (IJ (DoUCHA 1979) according toformula: L - II

ln LilII =

The va1ues of irradiance are expressed in W. m-2 PhAR. The incident (IJ andtransmitted radiation (IJ were measured by the phytoactinometer devised andconstructed in the author's laboratory (Tøeboò, Czechoslovakia) by Š. KUBfN

(KUBfN 1971).


Benzopyrene treahnent

Stock solution of 100 !tg BP per ml of acetone was used for dosage of BP into theexperimental cultures. Acetone was also added to the controls in these experi-ments. The final concentration of acetone in experimental cultures was kept farbelow its toxic level which was found to be 7.5 g .1-1 (BRINGMAN & KUHN 1980).Even at a relatively high concentration (5 !tg. ml-I), BP was neither crystallizedagain nor adsorbed by the glass wall of the culture vessels. Vigorous stirring ofalgal suspensions in the vessels guaranteed a homogeneous distribution of BP evenif it was not completely solubilized.

Determination of HP adsorption velocity

Aliquote parts of algal suspension were centrifuged and the BP concentfation wasestimated in both nutrient solution (supematant) and algal cells (sediment).

a) Nutrient solution. The extraction and assay of BP wa;carried aut.according to BORNEFF & KNERR (1959) by the method of adsorption chromatogra-phy with alkaline AlzO3 using benzene for elution. The absorbance of the eluatemeasured at 296 nm was used to determine BP concentration.

b) Algal cells. BP was extracted 3 times by methanol at 74°C and assayedaccording to BORNEFF & KNERR (1959).

Results

Stability of HP in solution under tbe experimental conditions used

No detectable instability or conversion of BP was found in pure nutrient solutionkept under either dark or light conditions for 6 days (Fig. lA). Furthermore, theinhibitory effects were the same in cultures with freshly added BP as in thosegrown in a solution with BP which had been illuminated for 3 days prior to startingthe experiment (not shown in the Figure).

RP adsorption by ceUs in suspension

The variance of BP content in both the cells and the nutrient solution was followedfor 6 days of cultivation in light (30 W. m-2) or under dark conditions. Exponen-tial decrease of BP amount in nutrient medium, even if applied at a high

concentration (5 !1g. ml-I, i. e. 10 pg per cell), was found in both variants(Fig. 1 b). More than 50% of the BP applied was adsorbed by the cells within 30minutes and about 80% was after 90 minutes of BP presence. No difference inadsorption rate occurred between the illuminated and darkened cells (Fig. lB).

286 VILÉM ZACHLEDER, ECKARD WIlTENBURG and SIBYLLE ABARZUA

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Fig. 1. Time variation of BP content in illuminated pure medium, in culture medium and inScenedesmus quadricauda cells. 5IA.g BP per ml was added into the pure medium (panel A)and into the cell suspension (panel B). The content of BP was deterrnined in bothilluminated (white symbols) and darkened samples (black symbols). Incident irradiance was30 W. m-2 and BP load 10 pg per cell.

The incident irradiance

The effects of BP were observed in cultures which were grown at different incidentirradiances (indicated in Fig.2). The BP (0.3 !tg' ml-1) was applied at thebeginning of the cell cycle and the cell number was kept equa! in both the controland the BP-treated cultures. Two para!lel experiments were carried aut with thecultures differing in cell concentration and, consequently, in load of BP per cell(120 fg per cell, Figs. 2A, 2B, 2C and 60 fg per cell, Figs. 2D, 2E, 2F).

In the cultures with the same cell density and the same BP load, the extent ofinhibitory effects of BP was affected by the incident irradiance (compare Figs. 2 A,B, C or 2D, E, F). A very early and strong inhibition of macromolecular syntheses(DNA, 'RNA, protein) occurred at the highest incident irradiance (200 W' m-2)combined with a sufficient BP load per cell (Fig. 2A). AIso the cell division wasblocked and the cells became bleached from the middle of the cell cycle.

With decreasing incident irradiance, the depression of macromolecular syn-theses occurred later in the cell cycle and to a lesser extent (Figs. 2B, 2c). At avery low irradiance, only a slight inhibition of both macromolecular syntheses and

cell divisions was observed (Fig. 2F).

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Fig. 2 A-F. The courses of RNA, protein, and DNA syntheses in the control and BP.treated synchronous cultures of Scenedesmus quadricauda grown at various incident andmean irradiances, and at two different cell culture densities and BP loads per celloBenzopyrene was added at the beginning of the cell cycle in concentration 0.3 Itg. ml-I: Thesame cell culture density was kept in both control (O, O, ~) and BP treated (8, 8, &)cultures by semi-continuous dilution. The values of the incident (IJ and mean (Ic) irradiancesare indicated in the individual panels. The courses of RNA (O, 8), protein (O, 8), andDNA (~, &) syntheses in control (fulllines, white symbols) and BP-treated (dashed lines,black symbols) cultures are shown. The cultures in the experiment illustrated in panels A, B,C differed from that in panels D, E, Fin the initial cell number (2.5 and 5 mil per cell) in BPload (120 and 60 fg BP per cell) and in culture density (absorbance at 750 nm 0.1 and 0.3),respectively. Glass cylinders (400 ml) served as culture vessels.

21 Archiv f. Hydrobiologie, Suppl.-Bd. 73

288 VILÉM ZACHLEDER, ECKARD WITTENBURG and SIBYLLE ABARZUA

Cell-culture density

From the results illustrated in Fig. 2 it is apparent that in addition to incidentirradiance it is the density that controls the extent oi BP inhibitory effects.Thereiore, the experiment was carried aut to examine the role oi this iactor in BP-cell interactions.

The same concentration oi BP per ml (0.2 lig) was applied to the experimentalvariants grown at the same incident irradiance (140 W. m-2) but differing in theiroptical density (from 0.05 to 0.5) and correspondingly in the cell number (from

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Fig.3. The courses of RNA, plotem, DNA and chlorophyn syntheses in benzopyrenetreated synchronous cultures of Scenedesmus quadricauda with a different cen culturedensity. Benzopyrene was added at the beginning of the cen cycle at a concentration of 0.2!tg. ml-I. The culture were grown at incident irradiance 200 W. m-2 PhAR. Meanirradiance was 120 W. m-2 PhAR. The absorbances at 750 nm about 0.5 (0),0.3 (O), 0.1(~) and 0.05 (+) were maintained by semi-continuous dilution of the cultures. Thecorresponding initial cen numbers were 7,4.8, 1.4, and 0.6. lQ6 ml-I, respectively, and theresulting BP loads per cen were 29, 42,132,333 fg BP, respectively. Glass cylinders (400 ml)served as culture vessels. Control curves are not shown bere, but they were sirnilar to thecurves indicated by (O).

300

200

100


0.6. 106 to 7 . 106 cells per ml). Virtually no effect was observed in the presence ofBP if the cell number was equal to or higher than 7 . 106 per ml (O.D750 = 0.5).The inhibitory effects of BP occurred below this threshold culture density (Fig. 3).The lower was the cell number the more extensive was the inhibitory effect of BPat any given incident irradiance. The most severe inhibitory BP effects onmacromolecular and chlorophyll syntheses were observed in cultures where thecell number was below 1.4.106 ml-l (O.D750 = 0.1). These syntheses were

inhibited completely after 4 hours of the cell cycle, and degradation of RNA,protein and chlorophylls occurred (Fig. 3). After a 10 hours' presence of BP, the

cells were bleached (see chlorophyll content, Fig. 3).

Mean irradiance

The above mentioned results indicate that both the cell culture density and theincident irradiance could play a role in the expression of the inhibitory effects ofBP. It was assumed that the mean irradiance might determine the;,extent of theinhibitory effects of BP. It would not be of a great importance, which combinationof the incident irradiance and the culture density would determine the meanirradiance per cell. The following findings supported this suggestion:

1) With the increase in a mean irradiance, a corresponding increase wasobtained in the inhibitory effects of BP. This rule applied irrespective of whetherthe mean irradiance increased as a result of an increase in the incident irradiance(Figs. 2A, 2B, 2C or 2D, 2E, 2F) or of a decrease in the cell density (Fig. 3).

2) The experiment was carried out with algal cultures differing in cell-culturedensity and grown at such incident irradiances that the same mean irradiance wasobtained in all experimental cultures. The BP concentrationper ml was chosen sothat the BP load per cell was constant. The results illustrated in Fig. 4 show thatthe same extent of BP inhibitory effects occurred in cultures growing at the same

mean irradiance.

Tbe concentration of RP per cen

It was observed lhal the cultures with a higher BP load per cell were moreinhibited even if grown at a lower mean irradiance (compare Figs. 2B and 2D or2C and 2E). Therefore, the effect of BP load should be considered as anotherfactor responsible for the extent of the BP inhibitory effects. Fig. 5 illustrates theresults of the experiment in which BP was applied, at a concentration of 0.3!tg' ml-l, to two cultures differing in their cell culture densities so lhal a differentBP load per cell was obtained. Both cultures were grown at the same meanirradiance. In accordance with the experiments described earlier, the same extentof inhibitory effects of BP was observed at the same mean irradiance when thesame BP concentration per cell had been applied to both cultures (Fig. 5). On the

290 VILÉM ZACHLEDER, ECKARD WI1TENBURG and SIBYLLE ABARZUA

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Fig. 4. Tbe courses of RNA and protein synthesis in control and BP-treated synchronouscultures of Scenedesmus quadricauda grown at different incident irradiances with differentcell culture densities but at the same mean irradiance and the same initial BP load per celloBenzopyrene was added at the beginning of the cell cycle in concentrations 0.1 !A.g, 0.3 and0.6!A.g per ml to obtain the same initial BP load (52 tg) per cell in all experimental cultures.Tbe courses of RNA and protein (marked in panels) in control cultures are designated bycircles (O, (), 8) and in corresponding BP-treated cultures by sq~res (O, [I, ').. Tbesame mean irradiance (about 115 W. m-2) was attained by the following combinations ofincident irradiances and cell culture densities (absorbances at 750 nm and initial cell numbersare given): 140 W. m-2, 0.1, 1.9.106 ml-I (O, O); 180 W. m-2, 0.3, 5.9.106 ml-I «), [I);210 W. m-2, 0.6, 11.5 . 106 ml-I (8, .). Plate-parallel cuvettes (1200 ml) served as culturevessels. Continuous dilution was applied to kept given optical densities.

Fig.5. The course of chlorophyll synthesis incontrol and HP treated cultures of Scenedesmusquadricauda grown at the same mean irradiancewith different HP concentrations applied percello Henzopyrene was added at the beginning ofthe cell cycle in concentrations 0.1 (O) and 0.3!tg. ml-I ([J, +) to obtain the same HP load52 pg HP per cell (O, [J) or three times higher158fg HP per cell (+). The same meanirradiance (about 115 W. m-2 PhAR) was ob-tained by the following combinations of incidentirradiances and cell culture densities (absor-bances at 750 nm and initial cell numbers aregiven): 140 W. m-2, 0.1, 1.9.06 ml-I (O, O,+) and 180 W. m-2, 0.3, 5.9. 1Q6 ml-I «), [J).Chlorophyll synthesis in the control cultures isillustrated by circles (O, (» and in HP-treatedcultures by squares (O, [J) or crosses (+).

- Plate-parallel cuvettes (1200 ml) served as cul-20 ture vessels. Continuous dilution was applied to

maintain the given optical densities.

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other band, the culture with a higher BP concentration was more affected thanthat with a lower concentration (Fig. 5).

Duration of 6gbt interval

As mentioned previously, a decreased mean irradiance results in a reduction of theinhibitory effects of BP. On the basis of this finding it may be assumed that noinhibitory effects of BP should appear in cells kept in the dark. This assumptionwas confirmed in ODe series of experiments when BP was added to subcultureswhich had been taken from a control culture at two-hour intervals and kept in thedark. Fig. 6 shows that the induction curves of the control culture coincided withthose obtained from samples kept in the dark in the presence of a relatively highamount of BP (200 tg) per cell, Irrespective of the cell-cycle time, the addition ofBP had no effect on the reproduction processes (DNA replication, nùtoses, celldivision) if the extemal energy supply was removed and the increase in RNA andprotein amounts prevented by subjecting the cells to dark treatmeQt.

Fig. 6. The potential of cells to divide if put inthe dark during the cell cycle in control syn-chronous culture of Scenedesmus quadricauda,in which HP had been present since the start ofthe dark treatment (period). (O, .) - quadru- _100plet induction curves, (I::., A) - octuplet induc- ~tion curves, (O, 1::.) represent the values for ~control culture. (., A) are valid for cultures ~which HP in concentration 5 !lg. ml-I was "O soadded to at the times of darkening. The cultures ~were grown at incident irradiance 200 W. m-2, ~mean irradiance 120 W. m-2, optical density at i750 nm was 0.15, cell number 2.5.10-6 cell per oml and HP load during dark intervals 200 fg percello

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In order to examine the assumption that the inhibitory effects of BP could beeffectively controlled by the duration of the light interval, BP was added at thebeginning of the celt cycle and the samples from both BP treated and controlcultures were successively put into dark at ODe hOUf intervals. The daughter cellsformed by the end of the control celt cycle were counted and the induction cuivesestimated (Fig. 7). The inhibitory effects of BP, demonstrated by decrease in thenumber of released daughter cells, increased with the duration of the lightinterval. It was mentioned before that the celt division may be completelysuppressed and macromolecular syntheses retarded in the cells grown in continu-ous light at a sufficiently high mean irradiance and BP load per cello However, incultures grown under the mentioned conditions, the inhibition of cell division caDbe partially or completely avoided by varyin~ duration the li~ht interval. As shown

VILÉM ZACHLEDER, ECKARD WI1TENBURG and SIBYLLE ABARZUA292

in Fig. 7, there is a period for which the capacity of BP-treated cells to divide in thedark increases with the progress of the cell cycle in a similar way as in the controlcultures. This means that within trus time BP affects neither the processes leadingto DNA replications, mitoses and cell division nor the growth processes.

100

Fig. 7. The potentia! of cells to divide if put inthe dark during the cell cycle in control syn-chronous cultures of Scenedesmus quadricaudaand those where HP was added at the beginningof the cell cycle. (O, 8) - quadruplet inductioncurves, (~,,) - octuplet induction curveso(O,~) represent the va!ues for control cul-tureso (8,.) are valid for the culture where003 !tg of HP per ml was adïed at the beginningof the cell cycleo The cultures were grown at themean irradiance 75 W o m-z PhAR (panel A)and 45 WO m-z PhAR (panel H)o Initia! cellnumber was 509 o 1Q6 cell per ml and HP load50 fg per cello Semi-continuous dilution wasused to keep optica! densities about 003 in a1lexperimenta! cultureso Plate-para1lel cuvettes(1200 ml) served as culture vessels.

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li the cells are darkened later in the cell cycle, the processes leading to celldivision start to be inhibited, and the capacity of cells to divide, even if darkened,decreases dramatically (Fig. 7 A, B). The period of time for which the inhibition ofcell division by BP caD be prevented by darkening the cells is dependent on themean irradiance. At high mean irradiance (75 W. m-2 PhAR), the inhibition ofcell división occurred during the first third of the cell cycle (the 5th hOUf of light(Fig. 7 A). At low mean irradiance (45 W. m-2 PhAR), however, this inhibitionoccurred during the second third of the cell cycle (the 11th hOUf of light) (Fig. 7 B).

These findings confirm that the growth rate (or synthetic rate) determined bythe light energy input, plays a decisive role in the control of BP inhibitory activity.It also follows from the above-mentioned experiments that for BP to becomebiologically active, a certain period of active growth of photosynthesizing algal

cells is required.

Time of RP application during the cen cycle

BP was applied at different times of the cell cycle and the subcultures were allowedto grow in light. A high mean irradiance (120 W. m-2) and high BP load per cell(160 fg) were chosen to promote the strong inhibitory effects of BP on the RNA,protein and DNA syntheses (Fig. 8) with BP applied at the start of the cell cycle.Fig. 8 shows that the extent of BP inhibitory effects became weaker if BP was


applied later in the cell cycle (e.g. in the 4th hOUf oflight); BP added stilllaterinthe cell cycle (8th hOUf) had only slight effects on macromolecular syntheses(RNA, protein and DNA).

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Fig. 8. The courses of RNA, protein, DNA, incontrol synchronous cultures of Scenedesmusquadricauda and those where BP added at diffe- 300rent times of the cel! cycle. Benzopyrene inconcentration of 0.3 Itg' ml-I was added at thebeginning (8--8), the 4th (8--8), and -the 8th ( ) hOUf of the cel! cycle. Times ~

Iof addition are represented by arrows with g 200

corresponding symbols. Individua! syntheses in :control (O - O) and BP-treated cultures ~(158 fg BP per cel!) are marked in the panels. SThe cultures were grown at incident irradiance ~ 100200 W. m-2 PhAR. Mean irradiance was 120 ~W. m-2 PhAR. Plate-para!lel cuvettes (2000 mlvolume) served as a culture vessels. The cul-tures were not diluted during the experiment.Initia! optica! density at 750 nm was 0.1 and cel! o

number 1.9. 106 cel!s per ml.

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Discussion

The results presented in this paper indicate that BP is withdrawn rapidly from thenutrient medium by Scenedesmus quadricauda cells. The accumulation capacity ofthe cells is surprisingly high (10 pg per cell), i. e., about 10% of cell dry mass). Thefact that algae may adsorb polycyclic aromatic hydrocarbons very efficiently wasreviewed by NEFF (1979) and MAy (1979). Moreover, GEYER et al. (1981) havefound an inverse log relationship between the water solubility of a wide range ofchemicals and their accumulation by the green alga Chlorella fusca. Thus, the lowwater solubility of BP seems to favour its adsorption by the algae from the nutrientmedium. Lipids of the cell membranes probably take part in adsorbing thelipophilic polycyclic aromatic hydrocarbons, as it was suggested by NEFF (1979).


Our findings indicate that the adsorption itself does not require any energy

supply because it occurs in both light and dark. By contrast, the BP transport from

the cell surface into the cell seems to be energy-dependent and light is required

under autotrophic conditions of cultivation. This is in a line with the observation

that BP adsorbed by cells in the light is washed out by organic solvents with greater

difficulties and to a lesser extent than is BP which was adsorbed in the dark

(WITfENBURG & ABARZUA, unpublished results).

The concentration per ml is usually considered to be the factor determining the

effects of many inhibitors. The present study indicates that BP does not belong to

such a group of inhibitors. The rapid and quantitative adsorption of BP from the

nutrient solution to the surface of cells may explain why the concentration of BP

per cell rather than per ml controls the extent of the BP inhibitory effects.

The present results provide evidence that the extent of BP inhibitory effects

can be efficiently controlled by the light conditions of algal cultQfe. On the other

band, our findings exclude any photoactivated reaction of BP under the given light

conditions. It seems, therefore, reasonable to assume that thelight has exclusivelyphototrophic function in BP - cell interactions and light - requiring processes are

involved in the conversion of BP into biologically active compounds. This assump-

tion is supported by the observation that BP must be metabolized prior to

becoming biologically active (ANDERSON 1979, PEZZUTO et al. 1978). Moreover,

the present results as well as those attained by other authors (GOUJON et al. 1980)

imply that a preceding synthesis of RNA and proteins is required for the BP

inhibitory activity to occur. These syntheses are required for a relatively long time

period in order to evoke BP inhibitory effects (ZACHLEDER et al. 1983) and their

rate is strictly regulated by the light input (ZACHLEDER & ŠETLfK 1982). These

findings indicate that the BP inhibitory activity in algal cells is controlled by the

growth rat.e (i. e. rate of RNA and protein syntheses) which is determined, in turn,

by the light energy input. This assumption is supported by our findings that the

rate of BP activation decreases in slowly growing cultures and no inhibitory effects

can be found below a certain threshold growth rate, even when very high

concentrations of BP are applied. Similarly, BP has no effect on cells kept in the

dark where further growth is stopped. However, the algal divisions were inhibited

even in the dark if the algae had been cultivated in the presence of BP at a

sufficiently high irradiance for a sufficiently long time. The stopping growth rate,

therefore, cannot reverse the BP inhibitions that occurred in the presence of BP

prior to darkening the cultures. As ODe reviews the literature there is no question

that BP itself has no effect on cell metabolism of mammalian and higher plant cells

and must be metabolized into biologically active compounds (ANDERSON 1979,

BORGEN et al. 1973, PEZZUTO et al. 1978, TRENCK & SANDERMANN 1978). As the

extent of inhibitory effects of BP is regulated by changing either the light

irradiance or the length of the period of illumination it may be assumed that the

same role is most probably valid in algal cells as well. Furthermore, a certain


photosynthetic work, given by the product of irradiance of the cell and the time ofits illumination, is apparently required for BP conversion into biologically active

compounds.

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Manuscript received Apri!, 15, 1985, accepted June, 13, 1985.

The authors' addresses:

Dr. VILÉM ZACHLEDER, CSc.,lnstitute of Microbiology,Czechoslovak Academy of Sciences,Dept. of Autotrophic Microorganisms,

Opatovický mlýn,CS-37981 Tøeboò, Czechoslovakia.

Prof. Dr. EcKARD WITfENBURG,Dr. ANNE-SIBYLLE ABARZUA,Wilhelm-Pieck-Universitiit Rostock,Sektion Biologie,Doberaner StraBe 143,nnR-?,OO R,,~t"rlc nnR

Documents

Factors controlling the inhibitory effects of 3,4-benzo(a)pyrene on the chlorococcal alga Scenedesmus quadricauda