Potent antileukemic activity of the novel cytostatic agent avarone and its analogues in vitro and in vivo.

Avarone and avarol are novel cytostatic agents which have potent antileukemic activity both in vitro and in vivo (mice). Cell culture experiments revealed that the cytostatic activity of these two compounds on L5178Y mouse lymphoma cells was 13- to 14-fold higher than that determined for HeLa cells and 40- to 43-fold higher than that for human melanoma cells. Nontumor cells (human fibroblasts and human gingival cells) were highly resistant against the two compounds. The inhibitory potency of avarone on L5178Y cells (50% inhibitory concentration, 0.62 microM) was significantly higher than the avarol activity (50% inhibitory concentration, 0.93 microM). Modification of the molecule at the quinone ring or the double bond in the terpenoid skeleton resulted in a significant loss of activity. In vivo studies with L5178Y cells in the ascites of mice confirmed the strong antileukemic effect determined in vitro. At doses of 10 mg/kg given i.p. once daily for 5 days to mice bearing approximately 10(8) leukemia cells, avarone was found to be curative in about 70% of the mice (20% for avarol). The optimal daily i.p. dose of avarone increased life span over controls by 146% when treatment was begun 1 day after tumor implantation and by 87% when treatment was delayed until day 8. Avarol, although active, was less effective. Based on the determined log10 kill values, avarone can be classified as a highly active and avarol as a markedly active cytostatic agent. The efficacy of the two compounds is also emphasized by the therapeutic index of 11.7 for avarone and of 4.5 for avarol. The two agents were determined not to be either direct mutagens or premutagens in the Ames test.

Inhibition of nuclear envelope nucleoside triphosphatase-regulated nucleocytoplasmic messenger RNA translocation by 9-beta-D-arabinofuranosyladenine 5'-triphosphate in rodent cells.

Nucleocytoplasmic translocation of polyadenylated messenger RNA is an energy-dependent process which is regulated by a nuclear envelope nucleoside triphosphatase; this enzyme was found to be stimulated by the 3'-terminal polyadenylic acid [poly(A)] tail of messenger RNA (Bernd, A., Schröder, H. C., Zahn, R. K., and Müller, W. E. G. Eur. J. Biochem., 129: 43-49, 1982). RNA efflux from isolated mouse lymphoma (L5178Y) cell nuclei is strongly reduced if 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP) is present in the transport medium. Half-maximal inhibition of RNA efflux occurs with 120 microM ara-ATP. Most likely, the inhibitory effect of ara-ATP is caused by inhibition of nuclear envelope nucleoside triphosphatase; this enzyme was found to be highly sensitive to inhibition by this antibiotic. The inhibition type of the nucleoside triphosphatase of rat liver nuclear ghosts is competitive with respect to ATP; the Ki:Km ratio was determined to be 0.27. Besides nucleoside triphosphatase, nuclear envelopes contain a protein phosphokinase modulating the affinity of pore complex laminae to poly(A). This enzyme was also found to be strongly inhibited by ara-ATP in a competitive way with respect to ATP (Ki:Km, 0.056) and could therefore also contribute to the overall inhibition of RNA transport. The polyadenylation of endogenous RNA by poly(A) polymerase(s) in intact rat liver nuclei as well as in nuclear matrices isolated from the same source was found to be markedly suppressed in the presence of ara-ATP. The inhibitions of both poly(A) polymerase activities (contained in whole nuclei or nuclear matrix bound) are of the competitive type with respect to ATP. In in vitro assays, nuclear envelope nucleoside triphosphatase is inhibited by microtubule protein. Of the 2 ATP-dependent enzyme activities associated with microtubule protein (cyclic adenosine 3':5'-monophosphate-dependent protein kinase and adenosine triphosphatase), only the kinase was slightly affected by ara-ATP. Cellular uptake of adenosine 5'-monophosphate and perhaps 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) is facilitated by a cellular membrane-bound 5'-nucleotidase. Our studies revealed that neither cleavage of ara-AMP nor inhibition of the enzyme activity by ara-AMP occurs. 9-beta-D-Arabinofuranosyladenine and ara-AMP represent neither direct mutagens nor premutagens as determined by the Salmonella-mammalian microsome mutagenicity test.

Aggregation of sponge cells. XX. Self-aggregation of the circular proteid particle.

In the extracellular space of the tissue of the sponge Geodia cydonium, circular proteid particles are found which carry as subunits the aggregation factor and a series of glycosyltransferases. Using the technique of velocity sucrose gradient centrifugation, the sedimentation coefficient (S020,w) of the particle-monosomes was determined to be 90. By means of the Svedberg equation a molecular weight of 1.3 . 10(8) daltons could be estimated. The monosomes aggregate in the presence of Ca2+ to higher complexes via disomes, trisomes, and pentasomes. The complexes can be redissociated by dodecyl sulfate but not by EDTA. During the Ca2+-mediated self-aggregation, the particles lose their biological activity with respect to their aggregation promoting function.

Cell aggregation of the marine sponge Geodia cydonium. Identification of lectin-producing cells.

A D-galactose-specific lectin, purified from the marine sponge Geodia cydonium, is present on the cell surface of mucoid cells, free choanocytes and choanocyte clusters, as revealed first, by the adhesion assay which is based on the formation of "rosettes" with erythrocytes, and second, by immunofluorescence studies. Using the same techniques no lectin could be identified on the surface of archaeocytes. Rosette formation was inhibited in the presence of 20 mM D-lactose as well as after preincubation of erythrocytes with purified lectin. Titration experiments in a hemagglutination assay showed that the highest level of extractable lectin (5% of the total protein) is found in mucoid cells, lower concentrations are determined in choanocyte clusters (0.07%), free choanocytes (0.05%) and archaeocytes (0.01%). Only the mucoid cells were found to synthesize lectin which is secreted and subsequently transferred to the cell surface of other cell types. As one consequence of the binding of the lectin to the cell surface of aggregation-deficient choanocytes or archaeocytes, the conversion of these cells to aggregation-susceptible ones is observed. These results support previous evidence that the lectin is involved in the reaggregation process of single cells in the homologous biological system.

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor.

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.

The potential of carp to bioactivate benzo[a]pyrene to metabolites that bind to DNA.

We have investigated the formation of DNA adducts in starved, fed and 5,6-benzoflavone-pretreated carp following i.p. administration of benzo(a)pyrene. 32P-postlabeling analysis of the liver DNAs showed the presence of one predominant (greater than 92%) adduct in all three groups. Cochromatography experiments revealed that the main adduct was identical to authentic BPDEI-dG (10 beta-(deoxyguanosin-N2-yl)-7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene). The formation of the adduct was evident as early as 1.5 h post-treatment and the levels increased steadily up to 7 days, reaching about 125, 110 and 102 attomole/microgram DNA in starved, fed and benzoflavone-pretreated carp, respectively. During this period, the benzo[a]pyrene-induced benzo[a]-pyrene monooxygenase activity increased from the uninduced, natural level of about 3 pmol/mg per min to levels of 35, 62 and 79 pmol/mg per min in starving, fed and 5,6-benzoflavone pretreated fish, respectively. A slow but steady formation of the diolepoxide-dG adduct was also observed in the liver DNA of carp following p.o. treatment. These results indicate that carp can biotransform polycyclic aromatic hydrocarbons such as benzo[a]pyrene to 'reactive' metabolites that bind to DNA.

A new type of hazardous chemical: the chemosensitizers of multixenobiotic resistance.

The purpose of this overview is to introduce the property of a new class of hazardous chemicals-the inhibitors of multixenobiotic resistance (MXR) in aquatic organisms, referred to as chemosensitizers. Aquatic organisms possess MXR, a mechanism similar to the well-known P-glycoprotein extrusion pump in multidrug resistant (MDR) tumor cells. MXR in aquatic organism moves from cells and organisms both endogenous chemicals and xenobiotics, including also some man-made chemicals. MXR in aquatic organisms represents a general biological first-line defense mechanism for protection against environmental toxins. Many chemical agents, the chemosensitizers, may after the function of this fragile mechanism. It is this new, MXR-inhibiting property, unrecognized as yet, that classifies these chemicals among top-rank hazardous water pollutants. The knowledge that the presence of one xenobiotic may block the pumping out of other xenobiotic(s), and hence accelerate their accumulation, may have important implications on environmental parameters like exposure, uptake, bioaccumulation, and toxicity. In this overview we present the evidence for the expression of MXR-phenotype in aquatic organisms, the demonstration of toxic consequences caused by MXR inhibitors, and the description of methods for measurement of concentration of MXR inhibitors in environmental samples.

DNA adducts in carp exposed to artificial diesel-2 oil slicks.

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by 32P-postlabeling in carp free-swimming in these tanks. 'Clusters' of lipophilic DNA adducts were detected, with five major and numerous minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to greater than 3000 amol/microgram DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (approximately 10,000 amol/microgram DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.

The effect of benzo[a]pyrene on sponges as model organisms in marine pollution.

The majority of the investigations were performed with the marine sponge Tethya lyncurium at concentrations of 2 X 10(-8) to 1 X 10(-11) g/ml of benzo[a]pyrene (BaP). Sea-pollution was characterized as BaP equivalent activity in the Ames test. Increased activity of ornithine decarboxylase (ODC) was observed when sponges were artificially exposed at polluted marine areas for 3 weeks. In contrast to the situation in higher animals no ODC induction of the fast type was observed. Mixed function oxygenases (MFO) were not detected in sponges nor could they be induced as in vertebrates. BaP was absorbed by Tethya and concentrated 30--60-fold. In live, but not dead, artificially perfused sponges [3H]- and [14C]BaP-radiolabeled became firmly associated with DNA, RNA and protein of the sponges. The association persisted in isolated fractions, in nucleotides, in nucleosides and in protein hydrolysates. The BaP binding ratio to DNA was found to be strongly correlated to the concentration of BaP. Light modifies BaP and thus enables binding. In the dark only very low association, if any, is observed. The possible consequences of these findings are discussed.

The chemosensitizers of multixenobiotic resistance mechanism in aquatic invertebrates: a new class of pollutants.

Mechanism of multixenobiotic resistance (MXR), identical to multidrug resistance (MDR) in tumor cells, has been found in aquatic invertebrates. The presence of this ATP-dependent membrane P-glycoprotein (Pgp) pump was confirmed by biochemical ('binding'), molecular (immunohistochemical, Western, Northern), physiological (verapamil-sensitivity) and toxicological (modulation of toxicity) methods. The inducibility of MXR in the presence of xenobiotics and its wide taxonomic distribution suggests its role as a general biological defense mechanism that rescues organisms by pumping potentially toxic xenobiotics out of the cells. Some xenobiotics, the chemosensitizers, can inhibit this defense mechanism. The presence of these MXR-inhibitors has important implications on environmental parameters like exposure, uptake, internal dose, bioaccumulation, response, synergism and toxicity. Such MXR-inhibitors, for example, enhance the accumulation of carcinogenic aromatic amines in mussel, with subsequent enhancement in production of their mutagenic metabolites, in induction of single strand breaks in DNA, and in induction of DNA-adducts. The property to inhibit defense mechanism of organisms classifies MXR-inhibitors among top-hazardous environmental chemicals. Therefore, we measured the concentration of chemosensitizers in water concentrates or sediment extracts as their potential to modulate the accumulation of fluorescent dyes in a cell-culture of NIH 3T3 mouse fibroblasts stable transfected with human MDR1 gene, or as the potential of native waters to decrease the efflux-rate of Rhodamine B from gills of mussels. We found significantly higher concentrations of MXR-inhibitors in samples from polluted marine sites or from polluted rivers than in samples from corresponding unpolluted sites. These concentrations were able to enhance the accumulation of fluorescent dyes or carcinogenic aromatic amines in clams, mussels, snails and sponges exposed to these xenobiotics, demonstrating the ecotoxicological relevance of MXR-inhibitors present in polluted waters.

High mutagenic potency of several polycyclic aromatic hydrocarbons induced by liver postmitochondrial fractions from control and xenobiotic-treated immature carp.

The metabolism of carcinogens in fish was examined by measuring the activation of different polycyclic aromatic hydrocarbons (PAH) by carp (Cyprinus carpio L.) liver post-mitochondrial fractions (S9) using the Salmonella typhimurium TA100 reverse mutation assay. For this study, 1 non-carcinogen, anthracene (AN), and 4 carcinogens, chrysene (CHR), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC) and 7,12-dimethylbenzanthracene (DMBA), were chosen. The bioactivating potency of the metabolic systems of carp pretreated with phenobarbital (PB), 3MC or Aroclor 1254 (ARO) were compared to uninduced carp liver. The results show that carp liver has the ability to metabolize carcinogenic PAH into mutagenic metabolites, which is enhanced when carp are pretreated with 3MC or ARO, but not with PB. A positive correlation between the induction of aryl hydrocarbon hydroxylase (AHH) activity in carp liver and the mutagenic potencies of CHR, BaP, DMBA and 3MC, has been observed. The bioactivating ability of carp liver S9 was compared with the ability of the same fractions from female Wistar rats (this study) as well as from Sprague-Dawley rats (literature data). When the mutagenic potencies of selected PAH had been normalized on the activity of BaP, the following order of mutagenic activities with S9 fractions from ARO-treated animals was obtained: (1) BaP (1) greater than DMBA (0.26) greater than 3MC (0.22) greater than CHR (0.05) greater than AN (0) for carp; (2) BaP (1) greater than 3MC (0.48) greater than CHR (0.31) greater than DMBA (0.16) greater than AN (0) for Sprague-Dawley rats; and (3) BaP (1) greater than 3MC (0.17) greater than DMBA (0.11) greater than CHR (0) = AN (0) for female Wistar rats. We conclude that carp and rats are very similar in their ability to activate carcinogenic PAH into mutagenic metabolites, which suggests that carp may be very susceptible to the carcinogenic activity of these compounds. According to our results from the mutagenicity study, as well as from the enzyme induction study, we propose the use of carp as a suitable model system for the study of chemical carcinogens.

Antimutagenic activity of the novel antileukemic agents, avarone and avarol.

The two antileukemic agents, avarone and avarol, were determined to be neither direct nor indirect mutagenic agents in the Ames microsomal test. Moreover, the two sesquiterpenoid compounds drastically reduced the mutagenic effect of benzo[a]pyrene in the same system. Subsequent enzymic studies demonstrated that avarone and avarol are powerful inhibitors of benzo[a]pyrene monooxygenase.

Increased genotoxicity of acetylaminofluorene by modulators of multixenobiotic resistance mechanism: studies with the fresh water clam Corbicula fluminea.

The presence of a 'multixenobiotic resistance' [MXR] mechanism in gills of the freshwater clam Corbicula fluminea was investigated. Western blot analyses of membrane vesicles from gills, applying antibodies to vertebrate P170 multidrug resistance (MDR) protein, revealed a 135 kDa immunoreactive protein. Verapamil caused a reduction of 3H-vincristine (3H-VCR) binding onto vesicles from clam. Exposure of clams to 3H-VCR in the presence of verapamil or staurosporine (STP) enhanced the accumulation of 3H-VCR over control values. Furthermore, clams were exposed instead to VCR, to a model carcinogen, 2-acetylaminofluorene (AAF), to determine the verapamil- and STP-dependent increase of single-strand breaks (SSBs) in DNA from gills of this organism. Verapamil caused no or little increase of SSBs induced by exposure to 0.01 or 0.10 microM AAF, respectively, as measured by the alkaline elution technique. In contrast, in the presence of STP a highly significant and dose-dependent enhancement of AAF-mediated SSBs was measured already at exposure to 0.01 microM AAF. These data indicate (i) that the clam C. fluminea is provided with a P-glycoprotein-like element of the MDR-mechanism, (ii) that this system can be poisoned by chemosensitizers such as verapamil and STP, (iii) the role of protein kinase C in the regulation of MXR function and (iv) the importance of the MXR modulators for the assessment of ecotoxicological effects of pollutants.

Fast ephemeral DNA damage upon BaP injection.

Injection of high concentrations of benzo[a]pyrene (10-125 micrograms BaP/g body weight) into young frogs (Xenopus laevis), fish (Gambusia affinis) or tissue culture cells (L 5178 Y) in a state of logarithmic growth causes alterations in DNA. In frogs these alterations reach a maximum at 60-90 min after application, then they decrease and become undetectable after 3 h. Within 8-12 days after the single BaP dose, a new wave of DNA alterations can again be detected. Parallel measurements of mixed function oxygenases (MFO) showed no short term activity changes within the first day. There was, however, an increase of activity starting on the 8th day following injection in frogs, and lasting until day 14. DNA alterations have been assessed by two methods. One is a modification of the alkaline filter elution method of Kohn et al. (1976) and is believed to determine the number of events leading to single strand breaks at alkaline pH. The other method determines the number of S1 nuclease sensitive sites in highly purified native DNA. Both results were found to be highly correlated. The effect does not seem to be caused by an impurity, unless an unknown, very minor, and extraordinarily active component is present. The effect in frogs is linked to nutritional status. Frogs fed a carbohydrate-rich, protein-poor diet do not give the fast response. The fast effect cannot be blocked by application of Actinomycin, Cordycepin, and Chloramphenicol injected either 30 min before or simultaneously with the BaP. Benzo(e)pyrene, a non-tumorigenic isomer of BaP, does not cause any effects at similar doses.

DNA damage by benzo[a]pyrene in the liver of mosquito fish Gambusia affinis.

Exposure of Gambusia affinis to water containing different concentrations of benzo[a]pyrene (BaP) causes an increase in benzo[a]pyrene monooxygenase (BPMO) activity which reaches a maximum on the second day. Concomitantly, the DNA is altered in such a way that nuclease S1-sensitive sites (SSS) become measurable. The size distribution of liver DNA treated with nuclease S1 in control fish shows two populations of DNA by length, with means of 30 X 10(6) and 60 X 10(6) Daltons, respectively. In fish treated with 100 ppb BaP, the population with longer molecules of DNA disappears and shorter molecules increase in number. This may be explained in terms of the introduction of an additional 0.31-0.46 DNA nicks per control DNA molecule caused by metabolically activated BaP derivatives.

Xenobiotic and endobiotic induction of mixed function monooxygenase in carp Cyprinus carpio.

3-Methylcholanthrene (3-MC) significantly induces aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in mature and juvenile Cyprinus carpio of both sexes. The degree of induction of AHH by 3-MC in mature fish is significantly lower than that in juveniles and induction in mature males is significantly higher than in mature females. Pretreatment with sex steroids (testosterone in males and estradiol in females) significantly reduces 3-MC-inducibility of AHH in both mature and juvenile fish, while gonadectomy in mature fish elevates AHH inducibility. AHH induction is slightly decreased by hydrocortisone and increased by metyrapone pretreatment in mature fish only. Gonadectomy further elevates metyrapone affected 3-MC inducibility of AHH in C. carpio.

Assessing consequences of marine pollution by hydrocarbons using sponges as model organisms.

Pollution has been assessed as a mutagenic activity determined by the Ames test, using radiolabelled benzo[a]pyrene (BaP) as a model pollutant. Experimental animals were sponges, mainly Tethya lyncurium from the Northern Adriatic and from the Pacific near Catalina Island, California, U.S.A. Changes in ornithine decarboxylase (ODC) activity (ODC; EC 4.1.1.17) and polyamine concentrations with and without pollution were observed. There is a slow rise in ODC activity during the course of three-weeks exposure and a fast increase of polyamine levels during the course of one day. Mixed function oxygenase (MFO; EC 1.14) activity could not be detected in sponges. There was a significant concentration dependent coupling of radioactive BaP derivatives (BaPD) to the macromolecular fractions; the highest in protein, X 1000 greater than DNA and X 500 greater than RNA. Coupling is light-mediated and drops to zero in the dark. However when activated microsomal fractions from fish, that had been exposed to high level polycyclic aromatic hydrocarbon (PAH) pollution are added, dark incorporation rises to significant levels which can be decreased by the MFO inhibitor 7,8-benzoflavone (BP). The question of possible absence of DNA repair in the sponges and some implications are discussed.

Activity of benzo[a]pyrene monooxygenase in fish from the Sava River, Yugoslavia: correlation with pollution.

Benzo[a]pyrene monooxygenase (B[a]PMO) activity in non-migratory fish from a given river segment is highly correlated to the recent pollutional history of that part of the river. The enzyme activity level can serve as a relevant measure for the harmful pollutant potential in aquatic ecosystems. Caged experimental fish exposed for about 10 days in river segments show BaPMO activity changes with the same predictive validity as that of the natural population.

DNA damage by PAH and repair in a marine sponge.

The sponge Tethya lyncurium from the Northern Adriatic has been used as an experimental species. A method is outlined for preparation of DNA which yields a highly purified DNA with a double-strand (ds) molecular weight of 25 M-dalton between single-strand (ss) breaks, which when properly damaged can be cut opposite to ss-breaks with nuclease S1. The molecular weights of the resulting ds-DNA pieces and their distribution has been evaluated by electron microscope photographs. Sponges exposed to benzo[a]pyrene (BaP) in the dark only incorporate BaP-derivatives (BaPD) in small amounts, if any. However, in the presence of light, derivatization to BaP derivatives enables effective coupling to occur, as shown previously (R.K. Zahn et al., 1981). Sponges were exposed to radiolabeled BaP in the presence of light. Coupling of BaPD to the DNA as well as the induction of ss-breaks were measured. Light-mediated coupling is concentration dependent from 0.01-20 ppb BaP with a correlation coefficient of r = 0.84. Under conditions of possible repair, ss-breaks completely disappear from sponge DNA in the course of three weeks while a substantial fraction of the BaP derivatives persists. Double label experiments show that substantial DNA synthesis occurs during this time. Pollution causes a decrease of the molecular weight of unnicked DNA, re-incubation in clean water an increase. A DNA species of 24 M-dalton seems to play a critical role. If its percentage in the DNA population drops below a critical level, recovery is not longer possible. DNA damage by PAH and repair in sponges seems to differ from that of most eucaryotes.

Potential multidrug resistance gene POHL: an ecologically relevant indicator in marine sponges.

Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic. Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system. Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans. It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system. Two cDNAs were cloned, one from the demosponge Geodia cydonium and a second from the hexactinellid sponge Aphrocallistes vastus. The cDNA from G. cydonium, termed GCPOHL, encodes a deduced polypeptide with a size of 34,591 Da and that from A. vastus, AVPOHL, a protein of a calculated M(r) of 34,282. The two sponge cDNAs are highly similar to each other as well as to the known sequences from fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae) and other Metazoa (from Schistosoma mansoni to humans). Under controlled laboratory conditions, the expression of the potential multidrug resistance gene POHL is, in G. cydonium, strongly upregulated in response to the toxins staurosporin (20 microM) or taxol (50 microM); the first detectable transcripts appear after 1 d and reach a maximum after 3 to 5 d of incubation. The relevance of the expression pattern of the G. cydonium gene POHL for the assessment of pollution in the field was determined at differently polluted sites in the area around Rovinj (Croatia; Mediterranean Sea, Adriatic Sea). The load of the selected sites was assessed by measuring the potency of XAD-7 concentrates of water samples taken from those places to induce the level of benzo[a]pyrene monooxygenase (BaPMO) in fish and to impair the multidrug resistance (MDR)/P-170 extrusion pump in clams. These field experiments revealed that the levels of inducible BaPMO activity in fish and of the MDR potential by the water concentrates are highly correlated with the level of expression of the potential multidrug resistance gene POHL in G. cydonium. This report demonstrates that the detoxification POH pathway, here mediated by the G. cydonium GCPOHL gene, is an additional marker for the assessment of the environmental load in a given marine area.