The genotoxicity of priority polycyclic aromatic hydrocarbons in complex mixtures

Risk assessment of complex environmental samples suffers from difficulty in identifying toxic components, inadequacy of available toxicity data, and a paucity of knowledge about the behavior of geno(toxic) substances in complex mixtures. Lack of information about the behavior of toxic substances in complex mixtures is often avoided by assuming that the toxicity of a mixture is simply the sum of the expected effects from each mixture component, i.e. no synergistic or antagonistic interactions. Although this assumption is supported by research investigating non-genotoxic end-points, the literature describing the behavior of genotoxic substances in complex mixtures is sparse and, occasionally, contradictory. In this study, the results of polycyclic aromatic hydrocarbon (PAH) analyses on freshwater bivalves were used to prepare realistic mixtures containing up to 16 PAHs. The SOS genotoxicity of the mixtures and each component were then assessed in an effort to evaluate the additivity of PAH genotoxicity. At nominal PAH concentrations above 1 microg/ml, observed genotoxic responses were far lower than those predicted under the assumption of additivity. At nominal concentrations below 0.75 microg/ml, differences are smaller and occasionally negligible, indicating that the genotoxicity of unsubstituted homocyclic PAHs is additive or slightly less than additive. Other researchers who have investigated the mutagenicity, carcinogenicity, and DNA binding activity of mixtures containing unsubstituted homocyclic PAHs have also reported additive effects. Therefore, the mutagenic risk posed by simple, well-characterized mixtures of priority PAHs can reasonably be estimated as the sum of the risks posed by the mixture components. Current data indicate that less-than-additive effects likely result from saturation of metabolic pathways needed to activate mutagenic PAHs.     

Induction of in vivo DNA adducts by 4 industrial by-products in the rat-lung-cell system

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), dibenzo[a,i]pyrene (DBP), and dibenz[a,h]acridine (DBAC) are by-products found in many industrial wastes and emissions. Workers in the related occupational settings are potentially exposed to these substances through inhalation. In the present study, induction of DNA adducts in vivo by these chemicals was investigated using ³²P-postlabeling analysis in the rat-lung-cell system. The potency of DNA-adduct inducing activity was also compared to that of two cytogenetic endpoints i.e., sister-chromatid exchange (SCE) and micronucleus formation. Via intratracheal instillation, male CD rats (6/group) were dosed 3 times with BA, DBA, DBP or DBAC in a 24-h interval. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, SCE and micronuclei. Results show that all 4 test compounds induced DNA adducts, SCEs, and micronuclei in the rat-lung cell in vivo and that the postlabeling DNA adduct assay detected genotoxic activity at lower dose levels than the two cytogenetic assays. These findings suggest that BA, DBA, DBP or DBAC are rat pulmonary genetoxicants and the DNA-adduct assay is more sensitive than SCE or micronucleus assays for detecting the pulmonary genotoxicity of these industrial PAHs in the in vivo rat-lung-cell system.     

Subcellular localization of proflavine derivative and induction of oxidative stress—In vitro studies

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem. 2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.     

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: Passive dosing versus solvent spiking

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source. It compensates for any losses and results in stable freely dissolved concentrations. Passive dosing using silicone O-rings was applied in the Ames II assay to measure PAH mutagenicity in strains TA98 and TAMix – a mixture of six different bacterial strains detecting six different base-pair substitutions – after metabolic activation by S9. Initially, 10 PAHs were tested with passive dosing from saturated O-rings, aiming at levels in the test medium close to aqueous solubility. Fluoranthene, pyrene and benzo(a)pyrene were mutagenic in both TA98 and TAMix, whereas benz(a)anthracene was mutagenic in TA98 only. The concentration-dependent mutagenic activity of benzo(a)pyrene was then compared for passive dosing and solvent spiking. With spiking, nominal concentrations greatly exceeded aqueous solubility before mutagenicity was observed, due to sorptive losses and limiting dissolution kinetics. In contrast, the passive dosing concentration-response curves were more reproducible, and shifted towards lower concentrations by several orders of magnitude. This study raises fundamental questions about how to introduce hydrophobic test substances in the Ames II assay with biotransformation, since the measured mutagenicity not only depends on the compound potency but also on its supply, sorption and consumption during the assay.     

Identification of purinoceptors in the isolated stomach and intestine of the three-spined stickleback Gasterosteus aculeatus L.

1. Adenine nucleosides and nucleotides were examined for pharmacological activity in isolated stomach and intestine from the stickleback Gasterosteus aculeatus L.2. Adenosine and its stable analogues all concentration-dependently relaxed carbachol-contracted stomach and intestine, with no significant difference in the potency of the analogues. Only 8-(p-sulphophenyl) theophylline inhibited the relaxant response to adenosine in both tissues; other adenosine antagonists such as 1,3-dipropyl-8-cyclopentylxanthine were not active.3. ATP, α, β-methylene ATP (α,β-MeATP) and 2-methylthio ATP (2-MeSATP) all caused concen- tration-dependent contractions of the stomach and intestine.4. In the stomach, the order of potency was 2-MeSATP >α, β-MeATP = ATP; the P_2γ-purinoceptor antagonist reactive blue 2 inhibited responses to ATP.5. In the intestine, the order of potency was α,β-MeATP > 2-MeSATP = ATP; reactive blue 2 did not affect responses to ATP, nor did prolonged incubation with α,β-MeATP.6. It is concluded that in both the stomach and intestine, adenosine is acting through a non-specific or undifferentiated P₁-purinoceptor. In the stomach, however, the P₂-purinoceptor appears to be analogous to the mammalian P_2γ-purinoceptor, and in the intestine, the receptor is more similar to the mammalian P_2x-subtype, although it was not susceptible to desensitization.     

Intrinsic mutagenicity of polycyclic aromatic hydrocarbons: A quantitative structure activity study based upon molecular shape analysis

The mutagenic potencies of 30 polycyclic aromatic hydrocarbons, (PAHs) were quantitatively related to the physicochemical properties of the parent structures. The goal of the work was to identify how much information regarding overall mutagenicity of PAHs reside in the unmetabolized structures. Quantitative structure activity relationships (QSARs) were established between measured mutagenic potency and two molecular properties: (1) ΔE, the difference in energy between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), and, (2) molecular shape, as measured by common overlap steric volumes between pairs of PAHs. Mutagenic potency can be predicted with an average error of about ±11% for these 30 PAHs. It appears that the physicochemical properties of a parent PAH dictate much of the mutagenic behavior ultimately realized through metabolic activation. Consequently, QSARs developed for the parent PAHs might serve as reliable empirical guides in ranking mutagenic potency.     

The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.     

Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs)

The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradation of PAHs in soils can be enhanced by employing surface-active agents. However, chemical surfactants are often recalcitrant and exert toxic effects in the amended soils. The effects of two biogenic materials as pollutant-mobilizing agents on the aerobic bioremediation of an aged-contaminated soil were investigated here. A soil historically contaminated by about 13 g kg(-1) of a large variety of PAHs, was amended with soya lecithin (SL) or humic substances (HS) at 1.5% w/w and incubated in aerobic solid-phase and slurry-phase reactors for 150 days. A slow and only partial biodegradation of low-molecular weight PAHs, along with a moderate depletion of the initial soil ecotoxicity, was observed in the control reactors. The overall removal of PAHs in the presence of SL or HS was faster and more extensive and accompanied by a larger soil detoxification, especially under slurry-phase conditions. The SL and HS could be metabolized by soil aerobic microorganisms and enhanced the occurrence of both soil PAHs and indigenous aerobic PAH-degrading bacteria in the reactor water phase. These results indicate that SL and HS are biodegradable and efficiently enhance PAH bioavailability in soil. These natural surfactants significantly intensified the aerobic bioremediation of a historically PAH-contaminated soil under treatment conditions similar to those commonly employed in large-scale soil bioremediation.     

Isolation and taxonomic affiliation of N-heterocyclic aromatic hydrocarbon-transforming bacteria

The azaarenes (nitrogen-containing heterocyclic aromatic hydrocarbons) are products of incomplete combustion processes and thus are widely distributed with tar and oil products in the environment. Despite their adverse organoleptic, toxic, and carcinogenic characteristics, the biodegradability and fate of multi-ring azaarenes have received little attention. This work demonstrates the presence of genetically diverse azaarene-degrading bacteria in coal tar-contaminated soils. Thirty-eight bacterial strains able to transform the three-ring azaarenes, 5,6- and 7,8-benzoquinoline, phenanthridine, phenazine, or acridine, were isolated. Only seven of these strains grew in liquid medium on the specific azaarene compounds on which they were isolated using plates; and the rest transformed the azaarenes without growth. Taxonomic characterization by 16S ribosomal DNA sequencing revealed that our enrichment technique provided a diversity of 18 different azaarene-transforming bacterial species. Only a few strains were able to mineralize the homocyclic analogue, phenanthrene. Several of the isolates, e.g., Dyadobacter fermentans, Methylopila capsulata, and Agrobacterium tumefaciens, were related to genera relatively unknown with respect to the biodegradation of xenobiotic compounds. These strains can provide further information on the fate of azaarenes in the environment.     

The synergism between hydrocarbon pollutants and UV radiation: a potential link between coastal pollution and larval mortality

Coastal, benthic invertebrates with complex life history strategies are exposed to stage- and habitat-specific selective forces. In the coastal environment, benthic adults are exposed to polycyclic aromatic hydrocarbon pollutants (PAHs) due to their proximity to human activities (shipping, urbanization, and industrialization). Benthic invertebrates produce lipid-rich eggs or larvae that absorb PAHs from polluted estuaries and coastal waters. The larvae of many coastal invertebrates move offshore following release from benthic adults. During development in offshore waters, larvae of some species are exposed to relatively high levels of ultraviolet (UV) radiation. Marine organisms vary in their tolerance to PAHs and UV radiation. The purpose of this study was to examine the effects of the sequential exposure of the larvae of marine crabs to PAHs and UV radiation.Using laboratory experiments, the larvae of four crab species were exposed to PAHs and UV radiation. There was a significant synergistic effect of exposure to PAH (fluoranthene or pyrene) and UV radiation on larvae of the spider crab (Libinia dubia), the stone crab (Menippe adina) and the mud crab (Panopeus herbstii). Larvae of blue crabs (Callinectes sapidus) were exposed to PAHs and UV radiation in both laboratory and solar UV experiments. Significantly higher mortality occurred for C. sapidus larvae using either type of UV-artificial or solar.Larvae of coastal invertebrates with complex life history strategies are susceptible to the combined effects of PAHs and UV radiation. In this study, the exposure of crab larvae to PAHs and UV radiation resulted in mortality to crab larvae using laboratory and solar UV experiments. There were no effects on larval crab mortality due to PAH or UV radiation independently but mortality was as high as 100% when both factors were present.     

Embryolethality and induction of 7-ethoxyresorufin O-deethylase in chick embryos by polychlorinated biphenyls and polycyclic aromatic hydrocarbons having Ah receptor affinity.

The lethality and 7-ethoxyresorufin O-deethylase (EROD)-inducing potency of some individual polycyclic aromatic hydrocarbons (PAHs) and coplanar polychlorinated biphenyls (PCBs) in chick embryos were measured in order to compare the mechanisms of action of these compounds. In previous studies it was found that coplanar PCBs and certain PAHs have a high embryolethality in the chicken and that they induce embryonic EROD activity. Although the most potent PAHs were almost as embryolethal as the PCBs when injected into hens' eggs 72 h prior to measurement, they were considerably less potent EROD inducers. In the present study, three coplanar PCBs (3,3',4,4'-tetrachlorobiphenyl (TCB), 3,3',4,4',5-pentachlorobiphenyl (PeCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (HCB)) and four of the most toxic PAHs (benzo[a]anthracene (BaA), benzo[k]fluoranthene (BkF), indeno[1,2,3-cd]pyrene (IP) and dibenzo[a, h]-anthracene (DBahA] were administered to chick embryos in different ways, including co-administration. Additive embryolethality was found when BkF and PeCB were co-administered as well as when BaA and DBahA were given simultaneously. The PAHs were more effective as EROD inducers when injected on day 9 (24 h prior to measurement) than when injected on day 7 (72 h prior to measurement). The opposite was found for PeCB and HCB, whereas no difference in potency was noted when comparing TCB injected 24 and 72 h before EROD determination. These substance-related differences were probably due, at least partly, to differences in biotransformation rates. EROD activities found after treatment with high doses of BkF, IP, or DBahA on day 9 were similar to those measured after treatment with PeCB in doses high enough to give maximal induction. Co-administration of high doses of BkF and PeCB did not further increase the activity, indicating that the PAHs and coplanar PCBs induce EROD to a common maximal value. To decrease the influence of metabolization of the PAHs on their EROD-inducing potency, EROD was determined early in development (day 8) and soon after treatment (24 h) in one experiment. In that experiment, the PAHs proved to be only a few times less potent EROD inducers in relation to their embryolethalities compared with the PCBs. The results of the present study, a previously observed similarity in pathology between chick embryos treated with PAHs and embryos treated with coplanar PCBs, and the fact that the most toxic PAHs also are the most avid Ah receptor binders suggest that the coplanar PCBs and the PAHs largely exert their toxicity in chick embryos via an Ah receptor-mediated mechanism. The differences between the compounds in their EROD-inducing potency/embryolethality ratios could probably be explained by their different rates of biotransformation.     

Structure-function relationships of somatostatin analogs

Objective of peptide chemistry has always been the production of analogues for clinical application. Advantages sought over natural peptides are (a) reduced molecular size; (b) prolonged biological half-life, and (c) enhanced specificity. After elucidation of the active core of somatostatin a number of analogues have been synthetized. Among them SMS 201-995, an octapeptide, was selected for further development because of its high potency and prolonged plasma clearance. Procedures extending the duration of action of somatostatin derivatives such as enhancement of lipophilicity and amino acid substitution are described, and factors influencing the specificity of such substances are succinctly analyzed.     

Synthesis,Antitubercular Activity and Mechanism of Resistance of Highly Effective Thiacetazone Analogues

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the β-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.     

The synthesis and evaluation of thymoquinone analogues as anti-ovarian cancer and antimalarial agents

Thymoquinone (TQ), 2-isopropyl-5-methyl-1,4-benzoquinone, a natural product isolated from Nigella sativa L., has previously been demonstrated to exhibit antiproliferative activity in vitro against a range of cancers as well as the human malarial parasite Plasmodium falciparum. We describe here the synthesis of a series of analogues of TQ that explore the potential for nitrogen-substitution to this scaffold, or reduction to a hydroquinone scaffold, in increasing the potency of this antiproliferative activity against ovarian cancer cell lines and P. falciparum. In addition, alkyl or halogen-substituted analogues were commercially sourced and tested in parallel. Several TQ analogues with improved potency against ovarian cancer cells and P. falciparum were found, although this increase is suggested to be moderate. Key aspects of the structure activity relationship that could be further explored are highlighted.     

Correlation of the aquarium goldfish toxicities of some phenols,quinones, and other benzene derivatives with their inhibition of autooxidative reactions

Hydroquinone when added to the aquarium water was found to be about a hundred times more toxic than phenol, to goldfish (and to Daphnia magna), but is only about twice as toxic when injected into fish or mammals. Tertiarybutyl catechol shows a similar high toxicity in the aquarium, while the toxicity of catechol, resorcinol, and pyrogallol approaches more closely that of phenol. As the substances of high aquarium toxicity are known to inhibit many oxidative and polymerizing autocatalytic "chain reactions," rank correlations were tabulated between the recorded inhibitory potency of various substances in these processes, and their aquarium toxicity for goldfish. The correlation between aquarium fish toxicity and electric oxidation potential (P 0.09) is more than suggestive, and becomes still more so if explainable discrepancies are excluded. Antioxidant fat stabilizers show suggestive correlation with fish toxicity (0.20), and better with electric oxidation potential (0.10). The photographic reduction potential gives suggestive correlation with fish toxicity (0.20) and somewhat better with the oxidation potential (0.15). The gasoline induction period correlation is more than suggestive with the oxidation potential (0.099), but rather poor for fish toxicity (0.265). The rubber anti-aging potency gives only poor correlation (0.39) with fish toxicity. The reasons for these divergencies are not clear; they may perhaps be connected with the solvent properties of the substrate. As an example, Lea (p. 175) cites that 0.01 per cent of maleic acid prevents rancidity of fats, but is rendered ineffective by the presence of water. Taken by themselves, no one of the P values is entirely convincing of the relationships stressed in this paper. However, the consistent finding of relatively small values of P lends considerable weight to the hypothesis that these chemicals act in a related manner; and that the chemical activity of a substance may furnish useful suggestions of its biologic potency, perhaps more so than the chemical constitution as such. The aquarium toxicity, for goldfish is a convenient means of classifying the biologic potency.     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Variations in the C-unit of bedaquiline provides analogues with improved biology and pharmacology

Analogues of the anti-tuberculosis drug bedaquiline, bearing a 3,5-dimethoxy-4-pyridyl C-unit, retain high anti-bacterial potency yet exert less inhibition of the hERG potassium channel, in vitro, than the parent compound. Two of these analogues (TBAJ-587 and TBAJ-876) are now in preclinical development. The present study further explores structure-activity relationships across a range of related 3,5-disubstituted-4-pyridyl C-unit bedaquiline analogues of greatly varying lipophilicity (clogP from 8.16 to 1.89). This broader class shows similar properties to the 3,5-dimethoxy-4-pyridyl series, being substantially more potent in vitro and equally active in an in vivo (mouse) model than bedaquiline, while retaining a lower cardiovascular risk profile through greatly attenuated hERG inhibition.     

Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.