Chloramphenicol: Magic bullet or double-edge sword?

Mutation Research, 196 (1988) 1-16

1

Elsevier MTR 07250

Chloramphenicol: magic bullet or double-edge sword? Herbert S. Rosenkranz Department of Environmental Health Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Received 17 November 1987) (Accepted 14 December 1987)

Keywords: Chloramphenicol; Chromosomal effects, of chloramphenicol, in somatic cells; CM; Hemophilis influenza; Lack of mutagenieity

Summary The genetic and genotoxic potentials of chloramphenicol are reviewed and analyzed. Although this widely used antimicrobial agent appears to cause chromosomal effects in somatic cells, in view of the consistent absence of other genetic effects, these cytogenetic abnormalities are ascribed to non-genotoxic causes. It is pointed out that despite its widespread use in human medicine, chloramphenicol has not been systematically tested for genotoxicity.

Chloramphenicol (CM, D-(-)-threo-chloramphenicol, CAS No. 56-75-7, Fig. 1) is a broad spectrum antimicrobial agent that is used to threat a number of human bacterial, rickettsial and chlamydial infections (Goodman and Gilman, 1975). Recently, as a result of the emergence of ampicillin-resistant Hemophilus influenza strains, CM has also been used increasingly to treat H. influenza meningitis, a life-threatening disease primarily of pediatric populations. In spite of its widespread use in human medicine, CM is not without side-effects including a reversible myelosuppression and a rare, but frequently fatal, aplastic anemia occurring at the rate of 1/40000 following oral administration of this antimicrobial agent. CM is a bacteriostatic agent (Brock, 1961) which means that it does not kill exposed bacteria, but

rather arrests their growth and permits the host's specific and nonspecific defense mechanisms to dispose of the invading microorganisms. Obviously, if the defense mechanism is impaired, discontinuation of CM therapy may result in a recurrence of bacterial proliferation and associated clinical symptoms. CM is a specific inhibitor of bacterial protein synthesis. It binds to the large subunit of the bacterial ribosome thereby blocking peptidyl transfer (Davis et al., 1980). However, exposure of eukaryotic systems to elevated levels of CM will result in inhibition of specific macromolecular

o~-Correspondence: Dr. Herbert S. Rosenkranz, Professor and Chairman, Department of Environment Health Sciences, School of Medicine, Case Western Reverse University, Cleveland, OH ,14106 (U.S.A.).

cg~J

~-~c8 c C~

Fig. 1. Structural formula of chloramphenicol.

0165-1110/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

~)CL;a

syntheses and of normal cell replication, as well as more general toxic effects. In humans, CM is rapidly absorbed from the gastrointestinal tract with a half-life of 1.5-3.5 h. It is well distributed in body fluids including the cerebral spinal fluid, brain, bile and milk. CM passes through the placental barrier and is inactivated by glucuronyl transferase. The resulting glucuronide is excreted in the urine. CM is available as the palmitate or the sodium succinate. Obviously, CM is a very useful as well as powerful drug which must be used with caution, i.e., it should only be administered when there is no alternate antimicrobial agent available and hematologic examinations of chronically treated individuals must be performed periodically. Evaluating the genotoxic properties of CM presents a real challenge as the substance is widely used in human antimicrobial therapy. CM is associated with a number of sequellae involving primarily the blood-forming system (see above) and these must be separated from possible genotoxic effects. The International Agency for Research on Cancer has classified CM in Group 2B (i.e., limited evidence of carcinogenicity in humans) (IARC, 1982). Additionally, CM is a nitroaromatic chemical, and as a class, nitroarenes are associated with mutagenicity and carcinogenicity (IARC, 1982; Klopman et al., 1987; Popp and Leonard, 1984; Rosenkranz, 1987; Rosenkranz and Howard, 1986; Rosenkranz and Mermelstein, 1985). Finally, chloramphenicol, as the name indicates, contains chlorine, a situation usually associated with environmental xenobiotics and not agents of human therapy. The study of CM at the molecular, cellular and organismic level is complicated by the fact that CM is an inhibitor of cytochrome P-450 isozymes (Halpert, 1982; Halpert et al., 1985; Miller and Halpert, 1986). In addition in bacteria, it is a potent inhibitor of protein synthesis while in eukaryotic cells it preferentially inhibits mitochondria. Additionally, as mentioned earlier, it exerts systemic toxicity. The common consensus regarding the basis of the biological activity of nitroarenes, of which CM is a member, iovolves the sequential reduction of the nitro function to nitroso and N-hydroxylamino derivatives and finally to arylamines. While

the arylamines are considered inactive, when compared to the nitro parent compound, both bacterial and mammalian enzymes can reoxidize them to the corresponding arylhydroxylamines. To cause mutagenicity a n d / o r carcinogenicity, the arylhydroxylamines may be transformed, nonenzymatically, to the corresponding arylnitrenium ions. However, because some N-hydroxylamine derivatives may not rearrange to the arylnitreniums spontaneously, the formation of an O-ester, e.g., O-acetyl derivative, may be an obligatory intermediary step and indeed, acetyl CoA-dependent transacetylases have been described recently in both bacterial and mammalian systems (DeFrance et al., 1985; Filer et al., 1985, 1986; Flammang et al., 1985; McCoy et al., 1982, 1983; Rosenkranz and Howard, 1986; Saito et al., 1985; Shinohara et al., 1986). With respect to CM, the presence of the corresponding amino derivative in the liver and kidney of guinea pigs receiving CM subcutaneously has been reported (Glazko et al., 1949). Additionally, both the cytosol and the microsome of rat liver have been shown to reduce CM to the corresponding amine (Fouts and Brodie, 1956). Similar effects have been ascribed to human-liver preparations (Salem et al., 1981). This reduction is not unexpected given the widespread distribution of nitroreductase activities (e.g., xanthine oxidase, alcohol dehydrogenase, DT diaphorase, aldehyde oxidase, etc.) in the cytosols and microsomes of many species. However, the detection of the amino analog of chloramphenicol suggests the presence of biologically active nitroso and hydroxylamino intermediates. The microbial flora is also capable of catalyzing this nitro reduction (Marca et al., 1982) and this is of relevance in view of the oral route of administration of this drug. In addition to nitroreduction, a microsomemediated oxidative pathway of CM metabolism which can result in cleavage of the side chain has been described (Morris et al., 1982). Some of the intermediates still posses an intact nitro function and hence can be reduced to additional, potentially mutagenic electrophilic species. Other metabolites of CM possess carbonyl functions, which, as a class, are known to be genotoxic. Finally, the microsome-mediated formation of halogenated alkane derivatives suggests the possi-

bility of additional products endowed with biological properties. Effect of CM on DNA in vitro

While CM has no effect on isolated DNA, electrochemically-reduced CM, presumably consisting of either the nitroso or the N-hydroxylamino derivative causes DNA damage, including strand distortion and chain scission as evidenced by decreases in the temperature of the Tm, the midpoint of the helix-to-coil transition profile. This change in Tm is accompanied by reduced viscosities and decreased molecular weights (Skolimowski et al., 1981, 1983). Additional studies with purified nitroso CM indicate (Murray et al., 1982) that the DNA damaging activity is due to a reduction product of nitroso-CM, presumably the corresponding N-hydroxylamino-CM. Indeed, N-hydroxylamino-CM can be formed from nitroso-CM in an environment of lowered oxygen tension (Eyer and Schneller, 1983). In many of its properties, CM resembles classical nitroarenes, i.e., ability of the nitroso intermediate to react with glutathione and with proteins (Dt~lle et al., 1980; Eyer, 1979; Eyer and Ascherl, 1987; Eyer and Schneller, 1983; Eyer et al., 1984; Klehr et al., 1985; Mulder et al., 1982) but not with DNA and reactivity of the corresponding N-hydroxylamino derivative or the derived arylnitrenium ion with DNA (Beland et al., 1983; Howard et al., 1983). On the other hand, the desnitro congener of CM, thiamphenicol, although endowed with antimicrobial activity, does not cause DNA damage (nor does it induce aplastic anemia, see below) (Skolinowski et al., 1983). Structural basis of the antimicrobial activity of chloramphenicol

CM, that is the D-(--)-threo isomer, is a broad spectrum antimicrobial agent that acts by virtue of its ability to interfere with ribosomal protein synthesis (see above). It should be noted that this activity does not seem to be dependent upon the nitro function per se as, for example, the L-(+)threo isomer is inactive. Bacteria insensitive to the antimicrobial action of CM are primarily resistant

as a result of inactivation of CM by an enzyme, chloramphenicol acetyltransferase, that leaves the nitro function intact. Moreover, desnitro-CM congeners may be endowed with antimicrobial activity (Manyan et al., 1975). Finally, neither nitroso-CM, N-hydroxylaminoCM nor the derived hydroxamates are more bactericidal than the parent CM. In fact, these derivatives demonstrate reduced antimicrobial activity (Corbett and Chipko, 1978), indicating that they are neither the proximate nor the ultimate antimicrobial agents. Of course, as with most antimicrobial agents, hundreds of derivatives of CM have been synthesized and tested for antimicrobial activity. However, a detailed study of structure-antibacterial activity is clearly beyond the scope of the present review. Suffice it to say that were CM found to present a mutagenic a n d / o r carcinogenic risk to humans by virtue of the nitro group, congeners of CM, devoid of this function, are available for antimicrobial therapy. Incidentally, as mentioned earlier, it would appear that it is the nitro function of CM that is associated with aplastic anemia, a rare, but frequently fatal complication, of CM therapy (Murray et al., 1982; Yunis et al., 1980) and that desnitro congeners, while not precipitating this side-effect, possess the same antimicrobial spectrum as CM. DNA damage in vivo: Salmonella and E. coil

A number of studies have shown that CM has no demonstrable effect on the stability of cellular DNA as determined by degradation to acid-soluble products upon prolonged exposure of E. coil to CM (Mullinix and Rosenkranz, 1971; Rosenkranz et al., 1971; Suter et al., 1978). Additionally, Kubinski et al., 1985, report that in E. coli there is no evidence of DNA modification following CM treatment, as determined by the absence of DNA binding to the treated cell. However, exposure of E. coli B / r and Salmonella typhimurium strains TA1976, TA1535 and TA100 to CM has been reported to result in the induction of DNA breaks as measured by sedimentation in gradients of alkaline sucrose (Jackson et al., 1977). This is somewhat surprising in view of the fact that such DNA damage is generally associated with bactericidal

agents acting as inhibitors of DNA synthesis (e.g., nalidixic acid, alkylating agents, ultraviolet light). CM, as discussed earlier, is a bacteriostatic agent which is a specific and reversible inhibitor of protein synthesis. It can be concluded, therefore, that this DNA damage is not associated with the bacteriostatic action of CM. Indeed, it is a property also exhibited by the L-(+)-threo isomer (Jackson et al., 1977) which is devoid of antimicrobial activity. It would thus seem that the DNA-damaging activity may be associated with the nitro function and that this effect could be dissociated from the antimicrobial, and therefore therapeutic, activity of CM. One of the possible explanations for the observation that bacteria exposed to CM exhibit evidence of DNA damage is that it reflects "spontaneous" breakage of DNA strands which require induced cellular DNA repair processes. Since such DNA repair may be dependent upon de novo protein synthesis, which cannot take place in the presence of CM, a specific inhibitor of protein synthesis, this could lead to apparent DNA damage. This illustrates the difficulty of dissociating the possible genotoxicity of CM from other effects. It should be mentioned that in this particular instance, the explanation of the inability of CM-treated cells to repair DNA damage may not apply, as the inactive isomer, i.e., the L-(+)-threo form, which presumably does not block protein synthesis, also induces DNA damage (Jackson et al., 1975). Additionally, it must be noted that CM induces the preferential synthesis and accumulation of plasmids even while cell division and new rounds of chromosomal DNA synthesis are inhibited (Clewell, 1972; Clewell and Helinski, 1972; Williams et al., 1982). This has two consequences of interest to the genotoxicity of CM: (1) The plasmid DNA which accumulates in the presence of CM contains segments of RNA (Blair et al., 1972; Williams et al., 1982) which are alkali-labile. Such molecules will exhibit modified sedimentation and chromatographic behaviors in the presence of alkali. These might be misinterpreted as reflecting single-chain DNA scission. (2) Many bacterial strains currently used for mutagenicity assays contain plasmids coding for inducible error-prone DNA repair enzymes, (e.g.,

plasmid pKM 101) and other DNA excision-repair functions. The amount of gene product made (such as nucleases) will be proportional to the number of copies of the plasmid present. Since, in the presence of CM, such plasmids are expected to accumulate, this may, in effect, lead to an apparent increase in DNA damage or modification. Indeed, some of the strains used to demonstrate DNA damage (Jackson et al., 1977) contain such plasmids, e.g., Salmonella typhimurium TA100 which carries plasmid pKM 101. The electron microscopic appearance of E. coli treated even for prolonged periods with CM does not support extensive DNA damage (Dworsky, 1974; Morgan et al., 1967). Such cells have pronounced nucleoids (i.e., DNA). Following removal of CM, accelerated nucleoid rearrangement occurs. This is unlike the situation seen following exposure of bacteria to specific DNA-damaging agents which is characterized by extensive alteration and even disappearance of the nucleoid material (Winshell, 1967). Moreover, the gross sedimentation properties of the nucleoid, as determined by sedimentation in gradients of neutral sucrose, did not show evidence of DNA breakdown following CM exposure (Dworsky, 1974). In vivo effects: B. subtilis

Exposure of Bacillus subtilis to even elevated levels of CM did not result in either single or double chain scission of the DNA as measured by sedimentation in neutral and alkaline sucrose gradients (Ohtsuki and Ishida, 1975). This is in contrast to the effect reported in E. coli and Salmonella (Jackson et al., 1977). On the other hand, CM did greatly enhance the DNA damage induced by another DNA-damaging agent (Ohtsuki and Ishida, 1975), again suggesting a confounding effect of CM, which presumably is related to its ability to block de novo protein synthesis and thereby possibly the induction of DNA repair enzymes. Induction of S O S functions

Exposure of bacteria to DNA-damaging agents results in a variety of responses designated the

TABLE 1 PAIRS OF DNA R E P A I R - P R O F I C I E N T A N D - D E F I C I E N T STRAINS IN

WHICH CHLORAMPHENICOL WAS TESTED

Bacteria

Relevant genotypes

Results

Reference

E. coil

pol A +/pol A -

-

Slater et al. (1971); Longnecker et al., (1974);

E. coil

E. coil E. coil E. coli

polA + lex A +/polA - lexAB/Bs_ 1 (excision repair-deficient) B/B/r (radiation-resistant) recA +/recA-

+

recB+ recC+/recB - recCrecA + recB+ recC+/recA - recB- recCuvrA + recA+/uvrA- recA-

+ +

Brem et al. (1974); Leifer et al., (1981a); Venturini and Monti-Bragadin, (1978); Nestmann et al. (1979); Levin et al., (1982); Simmon et al. (1977, 1978); Stissmuth (1980); Boyle and Simpson (1980) Venturini and Monti-Bragadin (1978) Shimizu and Rosenberg (1973) Shimizu and Rosenberg (1973) Suter and Jaeger (1982) Suter and Jaeger (1982) Suter and Jaeger (1982) Mitchell et al. (1980)

Salmonella

typhimuriurn Proteus mirabilis

uvr +/uvrB rec * hcr +/rec- hcr-

Bacillus subtilis

rec +/rec -

"SOS phenomena". These include filament formation, prophage and colicin induction and the induction of the sfi A gene product. These phenomena are normally blocked by specific repressors or by the iexA gene product. Following D N A damage, the recA gene product is activated and this in turn causes the SOS response by cleaving the specific repressor or the lexA gene product ( R a d m a n et al., 1977; Roberts and Devoret, 1983; Walker, 1984; Witkin, 1976). Recently, in order to improve the sensitivity of assays for the induction of SOS functions, the appropriate genes have been linked to genes which are expressed as the synthesis of enzymes which can be assayed colorimetrically, e.g., galactokinase, fl-galactosidase (for references, see Elespura, 1984). Before examining the effects of CM in these systems it should be noted that this antimicrobial agent inhibits the induction of the recA gene product (Satta et al., 1979). CM does not induce bacteriophage h in lysogenized E. coli ( h ) (Shimida et al., 1975). This does not appear to be due to a possible masking of the

Russell et al. (1980); Nader et al. (1981) Adler et al. (1976) Kada et al. (1972); Simmon et al. (1977, 1978); Karube et al. (1981); Sekizawa and Shibamoto (1982); Suter and Jaeger (1982)

phage-inducing effect as a result of CM's protein synthesis inhibiting properties, as CM does not block the phage-inducing properties of other genotoxic agents (Kozak and Dobrazanski, 1970). Unlike some frankly genotoxic agents (such as fl-propiolactone, M N N G and mitomycin C) and blockers of D N A synthesis (nalidixic acid, novobiocin), C M did not induce lysogenic strains of Staphylococcus aureus (Manthey et al., 1975). Nor does CM induce colicin (Ben-Gurion, 1978), an effect which also indicates the SOS response. In connection with the newer tests which link genes for error-prone D N A repair with enzymes such as fl-galactosidase, it must be recalled that if such genes are plasmid-borne, that they could yield false positive responses as a consequence of CM-induced plasmid amplification (see above) and resulting gene-dosage effects.

Preferential inhibition of DNA repair-deficient strains The preferential inhibition of bacterial strain defective in D N A repair was probably the first

genotoxicity assay developed. Such strains are preferentially inhibited by overtly genotoxic agents such as alkylating agents (Leifer et al., 1981b; Slater et al., 1971) as well as by inhibitors of DNA synthesis such as of DNA gyrase (e.g., nalidixic acid, novobiocin (McCoy et al., 1980)). In all such assays, except for E. coli strains deficient in recombinational activity (i.e., rec-), irrespective of the protocol and the bacterial strains used, CM inhibited both strains to the same extent, i.e., it gave a negative response (see Table 1). On the other hand, CM preferentially inhibited recA, recB recC and recA recB recC strains. However, these rec- strains also exhibited preferential toxicity towards other antibiotics known to affect structures other than the DNA (e.g., ampicillin, penicillin G, tetracycline). Accordingly, this effect probably does not reflect genotoxicity. Additionally, CM did not preferentially inhibit an E. coli uvrA recA strain (Mitchell et al., 1980). It is noteworthy that an E. coil K12 strain constitutive for the synthesis of DNA polymerase I and endonuclease I, did not display decreased sensitivity to CM when compared to the parental strain (Ahmad et al., 1980), which further confirms the lack of preferential inhibition of polA strains (Table 1).

Mutagenicity for bacteria CM, i.e. the D-(--)-threo isomer, is not mutagenie for E. coli as determined by reversion to either streptomycin-, proline- or tryptophan-independence (Hemmerly and Demerec, 1955; Mitchell et al., 1980). Additionally, CM was also found to be non mutagenic when tested for reversion to tryptophan-independence in the presence of $9 (Mitchell et al., 1980). However, in a forward mutational assay to L-azetidine-2-carboxylic acid resistance in E. coli WP2, CM did induce mutations. This effect was strongly associated with lethality and was also seen when bacteria were exposed to tetracycline, gentamicin and phosphonomycin (Mitchell et al., 1980). Since none of these agents have DNA as their target or affect DNA metabolism, it would appear that the observed mutagenic effect, which is inferred as resuiting from a deletion, is due to non-specific toxicity (Mitchell et al., 1980). Conflicting results were obtained when CM was tested in the Salmonella typhimurium reverse

mutational assay. While Mitchell et al. (1980), report CM to be weakly mutagenic for strain TA98, a number of investigators found it to be non-mutagenic for strains TA98, TA100, TA1530, TA1535, TA1537 and TA1538 (Brem et al., 1974; Jackson et al., 1977; McCann et al., 1975). In contrast, however, the L-(+)-threo isomer of CM was mutagenic for Salmonella typhimurium TA100 and TA1575, but not for TA98 (Jackson et al., 1977). On the other hand, an analog of D(--)threo-CM lacking the nitro function was nonmutagenie in these strains (Jackson et al., 1977). These data were interpreted (Jackson et al., 1977) as indicating (a) a role of the nitro function and (b) that the antimicrobial activity of the active isomer (i.e., D-(-)-threo) of CM masked the inherent mutagenicity of this agent. There are, however, some additional factors which make this a less than likely possibility: (1) Other nitro-containing antimicrobial agents are highly mutagenic (i.e., nitrofurans and nitroimidazole). (2) The highly sensitivity fluctuation test that was employed should have resolved the mutagenic and antimicrobial activities (see Green et al., 1977). (3) The pattern of responses of the inactive (L-( + )-threo) isomer, i.e., positive in both TA1535 and TA100 is unusual for a nitro-containing chemical. Most, if not all, such agents show no demonstrable mutagenicity in strain TA1535 as they require the presence of plasmid pKM101 (i.e., strain TA100) for expression of mutagenic activity in the standard Salmonella mutagenicity assay (Yahagi et al., 1976).

Yeast CM induced neither gene conversions nor mitochondriai mutations resulting in "petite colonies" in the yeast Saccharomyces cerevisiae strain D4 when tested in the presence or absence of an $9 preparation (Carneveli et al., 1971; Mitchell et al., 1980).

Plant systems Exposure of Arabidopsis to CM did not result in the induction of recessive lethal mutations (Miller, 1965). On the other hand, treatment of

barley root meristematic cells (Hordum vulgare) during S or early G phases resulted in the formation of "chromosomal fragments"; additionally, treatment during S phase also resulted in non-disjunctions (Yoshida et al., 1972; Yoshida and Yamaguchi, 1973). Treatment of Vicia faba with CM resulted in chromosomal aberrations ("anaphase with bridges", "anaphase with fragments") (Prasad, 1977). Exposure of the green alga Spyrogyra azygospora to CM caused a variety of chromosomal aberrations (Vedajanani and Sarma, 1978). Effect of CM on

Drosophila melanogaster

When injected into Drosophila melanogaster, CM was found not to induce sex-linked recessive lethal mutations (Clark, 1963). Additionally, when CM was present in food it induced neither sex-linked recessive lethal mutations nor dominant lethal mutations when administered to either male or female flies (Nasrat et al., 1977). Developmental effects of chloramphenicol

CM has profound teratogenetic effects on a number of echinoderms: the sand dollar (Deutch and Shumway, 1973) and several species of sea urchins (HagstrSm and LSnning, 1973; Lalli&, 1966). In one study on sea urchins, it was shown that this effect was dependent upon the presence of the nitro functions as well as of the D-(- )-threo configuration (Lallirr, 1966). Developmental anomalies were also observed when CM was added to the embryonic zebrafish (Anderson and Battle, 1967). Most of these teratogenic effects were accompanied by chromosomal a n d / o r mitotic anomalies. CM has also been reported to induce developmental abnormalities when administered to rats between the ninth and eleventh day of gestation (Dyban and Chebotar, 1971, quoted in IARC, 1976). Effect of chloramphenicol on cultured mammalian cells

Because of its inhibitory effect on cellular metabolism, it is not surprising that CM has

inhibitory effects on mitosis (see for example, Mittwoch et al., 1974). Additionally, CM affects viability of cultured mammalian cells (Yunis et al., 1980) as well as blocking DNA synthesis of cultured human bone marrow cells (Yunis et al., 1980). These latter two effects are even more pronounced when CM is replaced by nitroso-CM, suggesting that they may be involved in the aplastic anemia that occasionally follows treatment with CM. CM did not induce sister chromatid exchanges in cultured human lymphocytes (Pant et al., 1976a, b). On the other hand, while one study reports no effect of CM on the chromosomes of cultured human lymphocytes (Jensen, 1972), four reports describe the induction of chromosomal aberrations in such cultured cells (Goh, 1979; Mitus and Coleman, 1970; Pant et al., 1976a, b; Sasaki and Tonomura, 1973): gaps, breaks, fragments, secondary constrictions, translocations. CM induces chromosomal aberrations in cultured bovine and porcine lymphocytes (Babil6 et al., 1978; Qurinnec et al., 1975). However, CM concentrations which caused chromosomal aberrations in cultured human lymphocytes, did not induce such effects in cultured human diploid fibroblasts (Byarugaba et al., 1975). CM did not cause an enhancement in simian adenovirus-induced transformation of cultured Syrian embryo cells (Hatch et al., 1986). In vivo effects of chloramphenicoi

In view of the blood dyscrasias and myelotoxic effects induced by CM (see for example, Watson, 1977) the investigation of the possible chromosomal effects induced by this drug may be complicated. A further comfounding factor involves the ability of CM to affect chromosomal aberrations induced by known chromosomebreaking agents (Jensen, 1972) but not to significantly induce chromosomal effects in bone marrow of rats when administered alone (Jensen, 1972). On the other hand, when administered to mice it induced chromosomal aberrations in the bone marrow: chromatid-type aberrations (subchromatid and chromatid breaks) (Manna and Bardham, 1973, 1977).

Treatment of mice with CM did not result in an increased frequency of chromosomal anomalies (translocations) in spermatocytes and spermatogonia (Srhrn and Kocisova, 1974). CM did, however, increase the number of chromosomal aberrations induced by a polyfunctional alkylating agent (TEPA) (Srhm and Kocisovh, 1974). While CM did not cause dominant lethal mutations in mice (ICR strain) during the post-meiotic stage of spermatogenesis, it did have a dominant lethal effect on the premeiotic stage (Srhm, 1972, 1973). However, no such effects were reported by investigators using other mouse strains: I C R / H A (Epstein et al., 1972) or hybrid (101 × C3H)F (Ehling, 1971). On the other hand it was reported that following administration of CM to male Swiss albino mice, the F1 progeny exhibited a high incidence of chromosomal aberrations (chromatid-type breaks and others) (Manna and Roy, 1979). It has been suggested that in female mice (Beermann and Hansmann, 1986) CM interferes with oocyte maturation and the ordered chromosome segregation during the first meiotic division, thus increasing the risk of aneuploidy.

Cytogenetic effects in human The lymphocytes of patients exposed to therapeutic levels of CM have been reported to exhibit chromosomal breaks, gaps and fragmentations. Similar effects were seen when the lymphocytes of normal individuals were exposed in vitro to similar concentrations of CM (Mitus and Coleman, 1979). As mentioned earlier, CM may induce aplastic anemia; it is therefore perhaps significant that the lymphocytes derived from aplastic anemia patients do not exhibit an enhanced sensitivity to CM. However, such cells exhibit increased sensitivity to bleomycin (but not to mitomycin) when compared to lymphocytes of normal individuals (Morley et al., 1978). In view of the fact that CM-induced aplastic anemia may be a prelude to carcinogenicity (see below), these finding suggest that CM per se may not be the causative agent but that it may select for cells with increased sensitivity to certain DNA-damaging agents, such as bleomycin, a known genotoxicant (IARC, 1982).

Carcinogenicity Based upon case reports of leukemias in patients following CM-induced aplastic anemia, the International Agency for Research on Cancer classifies CM as possessing limited evidence for carcinogenicity in humans (IARC, 1982). With respect to animals carcinogenicity, IARC considers the data inadequate; there being only one study available suggestive to the induction of lymphomas in mice (Robin et al., 1981).

Interpretation of the results The broad spectrum of specific inhibitory effects as well as more generalized toxicity of CM in prokayotic and eukaryotic systems, make the study of the possible genotoxic effects of CM difficult. Certainly in view of the effects of CM on orderly DNA duplication and on the mitotic cycle, it is not too surprising that exposure to CM is associated with cytogenetic effects in plant and mammalian cells including possible evidence of nondisjunctions. However, as a whole what does the evidence suggest with respect to genotoxicity? Should we insist that a positive signal in a single type of assay is sufficient evidence for risk? Obviously the question is what risk are we referring to? Mostly we associate genotoxicity with risk of cancer causation. If this is indeed the criteria, than we could apply an objective procedure in which the test results are weighed in relationship to the performance of the tests with known carcinogens and non-carcinogens. The Carcinogenicity Prediction and Battery Selection (CPBS) method is one such procedure (Rosenkranz et al., 1986; Rosenkranz and Ennever, 1988a, b). The CPBS procedure has been described extensively on a number of occasions (Chankong et al., 1985; Pet-Edwards et al., 1985a, b; Rosenkranz et al., 1984, 1986). It provides an objective method for evaluating the potential genotoxic carcinogenicity of a substance based upon the results of short-term tests even when mixed (positive as well as negative) results are obtained. CPBS represents an application of Bayes' theorem which takes into consideration the performance characteristics of each assay vis-h-vis a panel of carcinogens and non-carcinogens. These performance characteris-

tics are d e r i v e d from the sensitivity ( a +) a n d specificity ( a - ) as d e f i n e d :

if the i n d i v i d u a l test result is positive

(3)

or a * = Number of carcinogens found positive in assay Number of carcinogens tested

0~+__,(1 - a ? ) Off

a - = Number of non-carcinogens found negative in assay Number of non-carcinogens tested

80+a

(1)

+ (1 - 8o+)(1 -

if the test result is positive, a n d 81+ = 0g(1 -

80+(1 - al + ) + (1 - Oo-)

- a,+) + ( - O , + _ l ) a f -

if the i n d i v i d u a l test result is negative

T h e basic C P B S e q u a t i o n s then allow the calculation of the p r o b a b i l i t y of c a r c i n o g e n i c i t y , 0 +, which for a single test T 1 is:

of =

0+i-I (1

(2) f

if the test result is negative. These e q u a t i o n s can also be used iteratively to calculate the results o f b a t t e r i e s w h e r e b y they a s s u m e the following forms:

0 +_ l a i + + (1 - 0 ,+_ , ) ( - a1 , - )

(4)

N o t e that the final value of 0 + is i n d e p e n d e n t of the o r d e r in which the test results are calculated. In the a b o v e e q u a t i o n s , the t e r m 00+ is the p r i o r probability of carcinogenicity ("prior" meaning b e f o r e testing) b a s e d u p o n the e x p e r t ' s i n t u i t i o n o r k n o w l e d g e f r o m such m e c h a n i s t i c c o n s i d e r a t i o n s as m e t a b o l i c p a t h w a y s , structure, electrophilicity, etc. In the a b s e n c e of such knowledge, Oo+ is assigned a value o f 0.5, which indicates that the c h e m i c a l has an e q u a l p r o b a b i l i t y o f b e i n g a carcinogen or a non-carcinogen. The assignment o f the 00+ value d o e s not, in fact, affect q u a l i t a tively the p r e d i c t i o n ( E n n e v e r a n d R o s e n k r a n z , 1986a, b) b e c a u s e the p r e d i c t i o n m a d e on the basis o f test results is c o m p a r e d with the 00+ value. Thus, s t a r t i n g with a 00+ value o f 0.5, after p e r f o r m i n g a series o f s h o r t - t e r m test, o n e m i g h t arrive at a p r e d i c t i o n o f c a r c i n o g e n i c i t y o f 0.93, which is a s u b s t a n t i a l increase in k n o w l e d g e a n d is therefore a c c e p t a b l e . O n the o t h e r h a n d , a p r e d i c t -

TABLE 2 SUMMARY OF SHORT-TERM TEST RESULTS Abbreviation

Test description

Result

a+

a-

pIA Prm

Escherichia coli DNA repair using POlA+ and POlA- strains Bacillus subtilis DNA repair using rec + and rec- strain Proteus mirabilis DNA-repair assay

-

0.836 0.906 0.836

0.5(7) 0.500 0.5(?)

Bsr EcW

E. coli WP2 reverse-mutation assay

Sty DR&

Salmonella mutagenicity assay Drosophila sex-linked recessive lethals

-

0.612 0.612 0.836

0.807 0.897 0.807

Cvt SCE Cbm VET Csp Csg Cle

In vitro chromosomal aberrations In vitro sister-chromatid exchanges In vivo bone-marrow cytogenetics Virus-enhanced transformation In vivo spermatocyte cytogeneties In vivo spermatagonia cytogenetics In vivo leucocytes cytogenetics

+ + +

0.890 0.890 0.836 0.890 0.333 0.667 0.836

0.667 0.667 0.5(7) 0.444 0.5(7) • 0.5(7) 0.5(?)I

a Due to low sensitivity and uncertain specificity, the performance of this test is unsatisfactory and will, therefore, not be included in the analysis.

10 TABLE 3 CPBS PREDICTION OF CHLORAMPHENICOL Battery

o,; 0~ 0f 0~' 04+ 05" 06+ 07' 08+ 094 01~~ O~ 01~

Prediction a

0.5oo

-

Cbm Cbm Cbm Cbm Cbm Cbm Cbm Cbm Cbm Cbm Cbm Cbm

~- Cvt + Cvt + Cvt + Cvt + Cvt + Cvt + Cvt + Cvt + Cvt + Cvt + Cvt

+ + + + + + + + + +

Cle Cle Cle Cle Cle Cle Cle Cle Cle Cle

+ + + + + + + + +

SCE SCE SCE SCE SCE SCE SCE SCE SCE

+ + + + + + + +

Sty Sty + Sty + Sty + Sty + Sty + Sty + Sty +

plA plA plA plA plA plA plA

+ + + + + +

DRL DRL DRL DRL DRL DRL

+ + + + +

VET VET VET VET VET

+ + + +

0.6257 0.8170 0.8819 0.5510 0.3713 0.1625 0.0380 0.0096 0.00l 8 0.0006 0.0003 0.0002

Bsr Bsr + PrM Bsr + PrM + EcW Bsr + PrM + EcW + C s g

" N o t e that the c o m p u t a t i o n p r o c e d u r e using Bayes' e q u a t i o n iteratively is c o m m u n a t i v e , i.e., the same result will be o b t a i n e d irrespective of the o r d e r in which the test results are c o m p u t e d .

ion of 0.55 might not represent a satisfactory increase in knowledge and the prediction would be considered inconclusive. In the above formulation of Bayes' equation, the values of et ÷ and ~ - are based upon the published reports of the Gene-Tox Program (Palajda and Rosenkranz, 1985) updated with results from the NTP and other relevant sources and further improved by cluster analysis (Pet-Edwards et al., 1985a, b). With respect to CM, the relevant tests and the responses obtained with CM are listed in Table 1. Using all twelve test results, which include 3 positive and 9 negative responses, and assuming a O0' value of 0.5, leads to the prediction (Table 3), that CM has only a 0.02% probability of being a carcinogen due to a genotoxic mechanism (i.e., a 99.98% probability of non-carcinogenicity). Moreover, the prediction is independent (Fig. 2) of the 00' between values of 0 and 0.9, which is indicative of a robust prediction (Rosenkranz and Ennever, 1988a, b). If we do not wish to dilute the three positive responses by so many negative ones, we could build a battery around the three positive responses (i.e., Cvt, Cbm and Cle). Such a battery could consist of Cbm, Cvt, Cle, SCE, Sty, PIA, and DRL. Such a battery would be considered risk averse, that is that the tests, except for Sty, are

sensitive but not necessarily specific. Such a battery is expected to minimize the number of carcinogens erroneously classified as noncarcinogens; however, it may falsely classify noncarcinogens as carcinogens (Ennever and Rosenkranz, 1986; Rosenkranz et al., 1986). Even when

Chloramphenicol 1.0

,

0

,

. O.O

,

,

0

,

.

.

.

.

~

1 .O Prior probability Fig. 2. The p r e d i c t e d lack of g e n o t o x i c carcinogenicity of C M as a function of the prior p r o b a b i l i t y using all of the analyzable test results (see T a b l e 2).

11

Chloromphenicol 10--

E O "U L O-

O.C 0.0

1.0 Prior probobility

Fig. 3. The predicted lack of genotoxic carcinogenicity of CM as a function of the prior probability using an abridged batter 3 which includes all of the positive test results. This batter5', consisting of Cbm. Cvt, Cle, SCE, Sty, plA and D R L can be considered risk-averse.

using such a battery, we still arrive at a prediction of non-carcinogenicity, i.e., O~- of Table 2 is 0.0380, i.e., 3.8% probability of carcinogenicity (96.2% probability of non-carcinogenicity) which by all currently used criteria is taken to indicate noncarcinogenicity (Ennever and Rosenkranz, 1987; Yander et al., 1987). Additionally the prediction for this battery is also nearly independent of the value assigned to the prior probability (Fig. 3), again indicating a robust prediction.

not been explored systematically. Rather, much of the data included in the present review were obtained incidentally and are derived from studies which used CM as a specific inhibitor of protein synthesis to elucidate the role of protein synthesis in genetic phenomena or as a negative control in genotoxicity assays. Obviously, were CM developed in the present scientific and regulatory climate, it would surely undergo much more extensive testing. In this connection, it must be pointed out that our search of the literature did not reveal results for many important genetic endpoints, e.g., gene mutations or the induction of unscheduled DNA synthesis in mammalian cells. Still, in view of its proven therapeutic benefits and the fact that in most countries its use is carefully monitored, it is doubtful that the risks of CM outweigh its benefits. Regarding its possible genetic and genotoxic effects, the only consistent findings are of chromosomal effects in somatic cells. These may well reflect the reversible CM-induced blockade of the cell cycle and of macromolecular syntheses. Accordingly, weighing all of the accumulated data, it would appear that CM per se does not present an unacceptable genetic or genotoxic risk.

Acknowledgements The help of the Environmental Mutagen Information Center in performing a literature search on chloramphenicol is gratefully acknowledged. This investigation was supported by the national Institute of Environmental Health Sciences and the U.S. Environmental Protection Agency.

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