Mutagenic effects of inhalational anesthetics

169

Mutation Research, 75 ( 1 9 8 0 ) 1 6 9 - - 1 8 9 © Elsevier/North-Holland

Biomedical Press

MUTAGENIC EFFECTS OF INHALATIONAL ANESTHETICS

JEFFREY

M. B A D E N * a n d V I N C E N T F . S I M M O N * *

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, and Anesthesiology Service, Veterans Administration Medical Center, Palo Alto, CA, and Department of Toxicology, SRI International, Menlo Park, C A , (U.S.A.) (Received 31 July 1979) (Revision received 24 October 1979) (Accepted 29 October 1979)

Contents Introduction ................................................. Physical and Chemical Properties .................................... Metabolism and Acute Toxicity ..................................... Carcinogenicity ............................................... Teratogenicity ................................................ Cellular Effects ............................................... Mutagenicity ................................................. Trichloroethylene .......................................... Fluroxene ............................................... Divinyl Ether ............................................. Other Anesthetics .......................................... Effects of Nitrous Oxide on Plants ............................... Summary and Conclusion ..................................... References ~ ............................................... .

.

169 170 172 174 176 178 179 179 181 182 182 184 184 185

Introduction William T.G. Morton of Boston is generally credited with the introduction of inhalational anesthesia into clinical practice. On October 16, 1846, at the Massachusetts General Hospital, he demonstrated the use of ether to produce a painless state for surgery. Within months, ether was used t h r o u g h o u t the world an an anesthetic. The introduction of chloroform quickly followed; in 1847 * Assistant Professor of Anesthesia, Stanford Un/versity School of Medicine and Staff Anesthesiologist VA Medical Center, Palo Alto, CA (U.S.A.) ** Microbial Genetics Depaztment, SRI International; Consulting Assistant Professor of Anesthesia, Stanford University School of Medicine. (Current address: Genex Corporation, 6 1 1 0 Executive Blvd, Rockville, MD 20852, U.S.A.) Address reprint requests to Dr. Badon.

170 James Simpson, an English surgeon and obstetrician, gave the first chloroform anesthetic to a human patient. Subsequently, chloroform was extensively used and became more popular than ether. However, reports of liver damage and other toxic effects following its administration led eventually to its abandonm e n t for human use. Nitrous oxide was introduced into clinical practice in 1863, 19 years after a failed demonstration of its anesthetic properties. Its popularity has never waned and it has remained the most widely used inhalational anesthetic. Ether, chloroform and nitrous oxide were the only available anesthetics until cyclopropane was introduced in 1933 and trichloroethylene in 1934. The first fluorinated anesthetic, fluroxene, was marketed in 1954. It was followed in 1956 by halothane which heralded the modern era of potent, nonexplosive, volatile anesthetics. Until recently, little t h o u g h t was given to possible adverse health effects from occupational exposure to anesthetics. Several early investigators reported that s y m p t o m s of generalized fatigue, headache and in some cases, heart disease were due to waste anesthetic gases [75,139]. Only in the last few years, however, have controlled epidemiologic studies been conducted [1,7,22,23,32,37, 38,44,83,84,110,114]. The most convincing finding has been an increased incidence of spontaneous abortion among female operating room personnel. Less consistent have been the findings of an increased incidence of congenital malformation among the offspring of exposed male and female operating room workers and an increased incidence of cancer among female operating room staff. There are still questions, however, concerning interpretation of these studies. Nonetheless, if these deleterious effects are real, exposure to inhalational anesthetics represents a considerable public health hazard. In the United States, about 50 000 hospital operating room personnel, including anesthesiologists, nurse-anesthetists and operating room murses and technicians, are exposed daily to waste anesthetic gases. Furthermore, surgeons, dental personnel and veterinarians and their technical assistants have a variable but sometimes heavy exposure. Exposure concentrations are extremely variable but may reach levels of a t least 50 ppm halothane and 5000 ppm nitrous oxide in unscavenged operation rooms [104]. It is estimated that the total number of exposed workers is 225 000 [104]. In addition, 20 million patients are anesthetized each year in 25 000 hospital operating rooms and 4.5 million patients are anesthetized by dentists [104]. Even if anesthetics have a low potential for causing long-term toxicity, one time exposure to high concentrations (e.g., 10 000 ppm halothane, 750 000 ppm nitrous oxide) of such a large population is likely to have a measurable effect. The mutagenic potential of inhalational anesthetics has important implications for long-term toxicity and is the main subject of this review. Carcinogenic, teratogenic and direct cellular effects are related topics and will be discussed briefly.

Physical and chemical properties The inhalational anesthetics are either gases or volatile liquids at room temperature and pressure. Of the gases, only nitrous oxide is currently in wide-

171

TABLE 1 PHYSICO-CHEMICAL PROPERTIES

Molecular formula

OF ANESTHETICS

Molecular w e i g h t

~ase8 Cyclopropane Nitrous o x i d e Ethylene

C3H 6 N 20 C2H 4

Volatile liquids Diethyl ether Chloroform Halothane Divin yl ether Trichloroethylene Fluroxene Methoxyflurane Enflurane Isoflurane

(C2H5)20 CHCI 3 CF 3 CHCIBr (C2H3)20 C2HC13 CF 3 CH2 --O--C2H 3 CHCI2CF2--O--CH 3 CHFCICF 2-O-CF 2 H C F 3 CHC1--O--CF2 H

Boiling point

Vapor pres. at 20°C

42.1 44 28

--33 --88.5 --103

74.1 119.4 197.4 70.I 131.4 126 165 184.5 184.5

36.5 61 50 28.3 87.5 42 104.8 56.5 58.5

Part. coef.

--

Oil/gas (Part. coef.)

11.2

--

1.4

--

1.28

425 160 243 553 60 286 23 180 250

50.2 265 224 58 720 56.8 930 98.5 94

Water/gas (volumes)

MAC for anesthesia (%)

0.22 0.44 0.08

9.2 I01 80

13 3.9 0.86 1.45 1.51 0.92 3.5 0.73 0.7

spread clinical use; the explosive agents ethylene and cyclopropane are n o w seldom used. The volatile anesthetics in c o m m o n use are either halogenated ethanes or ethers. Diethyl ether, divinyl ether, chloroform, trichloroethylene and fluroxene, are administered infrequently in the United States today because they are either flammable or are considered more toxic than those agents presently used. Diethyl ether and trichloroethylene, however, are still popular in some countries. The physical properties of anesthetics are shown in Table 1. One of these, lipid solubility, relates to both anesthetic action and toxicity and is of great pharmacological importance. Meyer [99] and Overton [ 1 0 9 ] were the first to observe that narcotic potency of an anesthetic is proportional to its oil/gas partition coefficient (Fig. 1). They suggested that narcotic action is due to the

:,00 <: ~

~

s oxi.

lo--

-6 ~: > 11,

, 0.1

1.0

I Cyclopropane ~1~ Fluroxene Enflurane~Ether ~,L Halothane "~hloroform "~Met,hoxyflurane 1~)

100

1000 5000

Oil/gas partitioncoefficient(37C) Fig. 1.

1.92 0.64 0.76 4 0.17 3.4 0.16 1.68 1.15

172 presence of the anesthetic dissolved in lipid cell membranes. Subsequent to Meyer and Overton's early work, several investigators have attempted to explain anesthetic action based on a disturbance of membrane function. For example, one theory b y Trudell [127] is based on lateral phase separation in cell membranes. Lipid solubility also determines the rate at which an anesthetic is eliminated from the b o d y ; the more lipid-soluble an agent, the longer the time required for its elimination. Because longer retention time increases availability for biodegradation, lipid solubility of a drug is an important factor in the accumulation of toxic and potentially mutagenic metabolites. Halogenation of aliphatic hydrocarbons decreases their volatility and flammability. Halogenation of ethers also decreases their water solubility and in some cases increases their lipid solubility. The greater the halogenation, the greater the decrease in volatility and flammability. Thus, the anesthetic fluroxene, which contains only 3 fluorine atoms is flammable, whereas the more highly halogenated ether anesthetics such as methoxyflurane (4 halogens) and enflurane (6 halogens) are not. The most stable halogen--carbon bond is formed with fluorine, followed in order b y chlorine, bromine and iodine. Carbon--halogen bonds are strengthened b y the presence of adjacent carbon--halogen bonds. Thus, polyhalogenated c o m p o u n d s have greater stability than monohalogenated compounds. Metabolism and acute toxicity Prior to the 1960's, inhalational anesthetics were regarded as classic examples of non-biodegradable b u t pharmacologically active drugs. It was believed that they were biochemically non-reactive and were taken up by the b o d y through the lungs and excreted unchanged b y the same route. However, in the last two decades, results of numerous studies have shown that all clinically used inhalational anesthetics are metabolized in vivo by the c y t o c h r o m e P-450 (mixed function oxidase) system. The enzymes of this system are situated primarily in the smooth endoplasmic reticulum (SER). The liver has a high concentration of SER and is the main site for the biotransformation of lipidsoluble c o m p o u n d s including anesthetics. Metabolism also occurs in the kidney, lung and to a lesser extent in other tissue. Oxidative dehalogenation and O-dealkylation are the principal chemical reactions involved in the metabolism of volatile anesthetics. In addition, epoxidation may occur in the biotransformation of anesthetics containing the vinyl moiety. A reductive pathway has been demonstrated for halothane. Although it is quantitatively less important than the oxidative pathway, it has significant implications for toxicity [33]. The metabolism of those anesthetics associated with specific organ toxicity has been closely examined. In particular, halothane, suspected of causing sporadic cases of postoperative liver failure, has been studied extensively. It is metabolized oxidatively to trifluoroacetic acid, bromine and chlorine as follows: F C1 I

I

F

Br

F-C--C--H I i

F O oxidation

I

U

> F--C--C--OH + Br- + C1I F

173 The results of studies by Van Dyke and Wood [133] in isolated perfused rat livers and by Cohen [31] using mouse whole-body autoradiography show that one or more metabolites of 14C-labeled halothane bind covalently to liver and other tissues. Trifluoroacetic acid is comparatively non-toxic and non-reactive and is unlikely to account for the binding. On the other hand, trifluoroacetaldehyde, which is a postulated intermediate o f the oxidative pathway, is highly reactive and could bind to tissue macromolecules. Of interest is the finding by Widger et al. [ 141], that the level of covalent binding in the liver increases under conditions of low oxygen tension. Based on their results, these authors suggested that halothane undergoes a reductive defluorination. In addition, Cohen et al. [33] isolated bromochlorodifluoroethane mercapturic acid from the urine of humans administered halothane. The presence of this metabolite also implies that a reductive defluorination pathway exists and that the reactive intermediate bromochlorodifluoroethylene is formed. This latter molecule is probably a p o t e n t alkylating agent [33]. Methoxyflurane, when administered in high doses, is occasionally followed by vasopressin-resistant, high-urinary-output renal failure [39]. This agent is metabolized to a greater extent than other anesthetics for t w o major reasons. First, the methoxyflurane molecule is comparatively unstable and can be attacked at either the dichlorocarbon or at the ether linkage as follows: oxidation

CH3 -O--CF2--CC12H

~ CH3--O--CF2--COOH + 2 C1-

oxidation

HCHO + HOOC--CHC12 + 2 F- HCHO + HOOC--COOH + 2 FSecond, it is more lipid-soluble than any other clinically used anesthetics and consequently is retained for a longer time in the body. Mazze et al. [96], and others found that inorganic fluoride (F-) produced as an end-product of methoxyflurane metabolism m a y reach sufficient concentration to be directly nephrotoxic. Oxalic acid is also a final metabolite of methoxyflurane biodegradation and if produced in high concentrations m a y contribute to renal damage [96]. Fluroxene is n o w seldom used, b u t is of interest because it is highly toxic to some species. A suggested pathway for its metabolism is as follows: CF3--CH2--O--CH=CH2

oxidation

* (CF3CH~OH) + CH2 = CHO {CF3CHO)

COs

CF3COOH Gion et al. [62], reported that the main end-product of fluroxene metabolism in man is trifluoroacetic acid which is n o t associated with much toxicity. In the rat, mouse, dog and other species, however, trifluoroethanol and trifluoroacetaldehyde are produced and are the likely cause of the extensive liver and kidney damage seen after fluroxene administration [27,69,79]. Chloroform, which is n o w rarely used as an anesthetic, is generally regarded as a classic hepatotoxin. Brown and Sagalyn [21] found that the liver damage produced by chloroform is dose-dependent and its toxicity and metabolism are increased by pretreatment of animals with the enzyme inducer, phenobarbital. Furthermore, Illet et al. [76], showed that the metabolic products of chloro-

174 form are covalently b o u n d to liver-tissue macromolecules, and that the degree of binding parallels the extent of liver damage. It is presumed that reactive free radicals or phosgene formed during chloroform's biodegradation are responsible for the liver changes [94]. Whatever the mechanism of chloroform's hepatotoxicity, the finding of metabolites covalently b o u n d to the liver and other organs m a y relate to long-term toxicity including potential mutagenic effects. Trichloroethylene (TCE) is of interest because it was the first inhalational anesthetic shown to be biotransformed. Furthermore, if used in closed circuit anesthesia with a CO2 absorber such as soda lime, toxic products including the p o t e n t nerve poison dichloroacetylene m a y be produced; paralysis of cranial nerves or even death have occurred when TCE has been accidentally used in this manner. Henschler et al. [72], suggested that TCE undergoes metabolism in vivo to trichloroacetic acid, trichloroethanol and monochloroacetic acid and that an epoxide and chloral hydrate are formed as intermediates. Banerjee et al. [14], showed that an unidentified metabolite of TCE b o u n d covalently to microsomal protein and salmon sperm DNA and that the degree of binding was correlated with t u m o r induction. The metabolic pathways of other anesthetics listed in Table 1 have also been studied. In general, no specific organ toxicity has been associated with their biodegradation and they will n o t be further discussed. A detailed review of the metabolism of volatile anesthetics and the implications for toxicity have recently been presented b y Cohen and Van Dyke [34]. Carcinogenicity Structural similarities exist between volatile anesthetics and human or animal carcinogens. For example, methoxyflurane, enflurane and isoflurane are alpha halo-ethers as are the carcinogenic b u t non-anesthetic chemicals bis(chloromethyl) ether, chloromethyl methyl ether and bis(~-chloroethyl) ether (Table 2) [92,131,137]. The anesthetics, halothane and chloroform are alkyl halides; methyl iodide, butyl bromide, b u t y l chloride and butyl iodide are from the same chemical class and are animal carcinogens [112]. Finally, the anesthetic and inductrial solvent, trichloroethylene is a halogenated alkene similar to the human [40] and animal [134] carcinogen vinyl chloride. Fluroxene and divinyl ether also Contain the vinyl moiety. These observations do n o t prove that anesthetics have carcinogenic activity b u t are of interest in the light of results from epidemiological surveys and animal experiments. In a nation wide retrospective survey of a b o u t 50 000 operating room personnel conducted in the United States by an Ad Hoc Committee of the American Society of Anesthesiologists [1], a 1.3- to 2.0-fold increase in cancer rate was noted among female members of the American Society of Anesthesiologists and American Association of Nurse Anesthetists compared to matched controls. No increase in cancer rate was seen among male operating room workers. However, the significance of the findings from this survey has been questioned [136]. It is of interest though, that Cohen [35], recently reported a doubling of the cancer incidence among 150 000 female dental assistants b u t not among 100 000 male dentists who work with inhalational anesthetics. In a separate survey, Corbett et al. [38], examined a small population

175 TABLE 2 STRUCTURAL FORMULAS OF SEVERAL KNOWN HUMAN CARCINOGENS AND THE INHALATIONAL ANESTHETIC AGENTS

Carcinogens

Inhalational anesthetics

H

H--C--CI

fl --I

F

I

el Chloroform

H

Methyl iodide

H

H

H H

Bis(~-ehloroethyl) ether

cl

Cl

H

H

Bis(chloromethyl)ether

F

H

Halothane ( f l u o t h a n e )

F

Isoflurane (forane) H I

C1 F

H

M e t h o x y f l u r a n e (penthrane)

fl -T-OT-H H

H

f[

f

CI F

F

C h l o r o m e t h y l m e t h y l ether

Enflurane (ethrane)

H

CI

C1

cc H

= \H

V i n y l chloride

C1

I I I I F Br

F--C-.-.C--H

CI

~.c / H/

~'Cl

Trichloroethylene

of 525 Michigan nurse anesthetists and suggested there was a higher incidence of cancer among this group. Not all authors, however, agree that the increased cancer incidence is real [ 1 3 5 , 1 3 6 ] . Furthermore, these studies do not prove that a cause and effect relationship exists between anesthetics and cancer; other factors may account for the results. For instance, Fink and Cullen [56], after a careful examination of all the data, suggested that stress among operating room personnel was as likely a factor in occupational cancer as was contamination by trace levels of the inhalational anesthetics. Other epidemiologic studies have been negative. In both retrospective and prospective surveys spanning 25 years, Bruce et al. [22,23], were unable to find a statistically significant increase above normal in the death rate among anesthesiologists due to malignancies. The statistics for this study were based on 652 deaths. Doll and Peto [44] reported that the overall death rate among 1252 male, English anesthetists, over 35 years of age, who were followed prospectively for 20 years was 93% and the death rate due to cancer was only 79% of the expected rate among physicians. Finally, a joint study by the American Society of Anesthesiologists and the American Cancer Society confirmed the findings of Doll and Peto [93] ; based on 637 deaths, the overall death rate of

176

male anesthesiologists was 84.1% of expected and death rate due to cancer was the same as among all physicians. In a brief critical review of the above published epidemiological studies, Vessey [135] concluded there was little convincing evidence that any form of cancer is an occupational hazard from working in the operating room. The authors o f the present review are unaware of epidemiological data concerning the cancer incidence of patients who have received one or more anesthetics. The results of animal carcinogenicity experiments have been variable and their interpretation has been questioned [13]. When administered in large dosages by oral gavage, chloroform produced liver cancer in B6C3F1 mice and renal tumors in male Osborne-Mendel rats [43], whereas trichloroethylene caused liver tumors in mice b u t n o t rats [103]. 50 animals were used in each treatment and control group. Although oral gavage is appropriate for studying dietary and therapeutic intake of chloroform or trichloroethylene, it is not clear if this route of administration is relevant to inhalational exposure of patients or to occupational exposure in the operating suite. Furthermore, when trichloroethylene was studied the agent administered contained 0.19% 1,2e p o x y b u t a n e and 0.09% epichlorohydrin, both known mutagens [88,97,118]. In addition, epichlorohydrin is a rodent carcinogen [132]. The presence of these contaminants, though in trace amounts, casts d o u b t on the significance of the trichloroethylene data. In the only positive study via inhalation of an anesthetic, Corbett [36] reported at increased incidence of liver tumors in male b u t n o t female Swiss/ ICR mice exposed to the experimental anesthetic, isofiurane. However, several confounding factors make the interpretation of this study open to question. For example, there were high levels of polybrominated biphenyls in the livers of isoflurane treated mice which m a y have contributed to the increased incidence of liver tumors. By contrast, a recent study by Eger et al. [51], which was co-authored b y Corbett, did not demonstrate an increased incidence of liver or other tumors in Swiss/ICR mice exposed to either isoflurane, halothane, enfiurane, methoxyflurane or nitrous oxide. The exposure regimen was similar to that originally used b y Corbett, although group sizes of a b o u t 200 animals were much larger. Also, Baden et al. [13], did n o t show an increased incidence of benign or malignant tumors in 161 Swiss/ICR mice administered a maximum tolerated dose (500 ppm) of halothane by inhalation for 18 months and sacrificed after 20 months. Finally, a study b y Coate et a1.[29], failed to demonstrate increased t u m o r incidence in groups of 50 Fischer 344 rats exposed by inhalation for lifetime to several subanesthetic concentrations of a nitrous oxide/halothane mixture. Teratogenicity A significant and consistent finding of several surveys has been a higher than expected incidence of fetal wastage among operating room personnel. In a survey of 303 Russian anesthesiologists, Vaisman [130] noted that 18 of 31 pregnancies ended in spontaneous abortions. However, there was no control group. Askrog and Harvald [6] reported a higher rate of spontaneous abortion among Danish anesthetists. Approx. 20% of 392 pregnancies starting after em-

177 p l o y m e n t ended in spontaneous abortion, compared to 10% of 212 pregnancies among the same group prior to operating r o o m employment. Cohen et al. [32], reported spontaneous abortion rates among 67 operating r o o m nurses and 50 female anesthetists of 30 and 38% resp.; spontaneous abortion rates in control groups of 92 female nurses and 82 physicians were 10%. In a study from the United Kingdom, Knill-Jones et al. [83], found the rates of spontaneous abortion, to be higher among 563 married, female anesthetists than among 828 married, female, non-anesthetist physicians. In a survey of 50 000 operating r o o m anesthesiologists, nurse anesthetists, nurses and technicians and 24 000 unexposed physicians and nurses, c o n d u c t e d in the United States, the incidence of spontaneous abortion was 1.3--2 times higher among exposed than among unexposed females [1]. Finally, a study b y Rosenberg and Kirves [114] confirmed the results of the other studies. Despite a negative survey b y Pharoah et al. [110], when taken together the above studies provide reasonable evidence for an increased risk of spontaneous abortion among female operating r o o m workers. They do n o t necessarily prove that anesthetic exposure is the cause. The above studies [1,6,32,83,110,114] and t w o others [37,84] assessed the incidence of malformation among the offspring of exposed females. The findings were less consistent than those for spontaneous abortion; negative results were obtained in all b u t t w o studies [1,37]. Other hazards such as abortion among wives and malformation among offspring of exposed males have also been investigated [1,6,84]. Again, there is little consistency of results among the studies and firm conclusions of possible hazards will have to await further studies. The effect of inhalational anesthetics on the reproductive processes of experimental animals has been the subject of many reports. This topic has been reviewed in t w o comprehensive publications by Ferstandig [54] and by NIOSH [104] and will n o t covered in the present review. However, several points are worth emphasizing. The vast majority of studies were with either nitrous oxide or halothane; other anesthetics received less attention. There were few studies on behavioral teratogenic effects of anesthetics. In general, studies were of t w o types. The first involved testing high concentrations of anesthetics at various times throughout pregnancy. In some cases, levels administered were even greater than those used clinically. The aim of these studies was to simulate the clinical situation in which patients receive general anesthesia during pregnancy. However, in the animal studies, the physiologic effects expected at high anesthetic concentrations were n o t controlled. Thus it is not possible to separate the adverse affects of such factors as hypoxia and hypotension from the direct effects of the anesthetics. In addition, chick embryos were used as the test animals in many of the studies, a model which is of questionable value for assessing the risk of chemical hazards in man. In the second type of experiment, the effects on reproduction of repeated doses of trace anesthetic concentrations were assessed. The aim was to parallel occupational exposure to waste anesthetic gases in the operating suite. Most studies gave negative results. However, the methods employed in these studies and the results obtained were quite variable. In addition, many of the studies were inconclusive because of lack of proper controls and incomplete examination of maternal and fetal tissues. Thus it appears that standardized studies

178 with adequate controls are still needed if the effects of anesthetics on reproductive processes are to be determined and comparisons are to be made among agents. Cellular effects The effect of anesthetics on cell division has been studied for more than one hundred years. Bernard [18], in 1878, demonstrated that ether vapor inhibited plant growth and cell division in seedlings. 5 years later, Martin [95] showed that seeds exposed to a gas mixture of 88% nitrous oxide/12% oxygen t o o k twice the time to germinate. In 1944, ()stergren [106] found a colchicine like mitotic (c-mitotic) effect when r o o t tips of Allium cepa were exposed to nitrous oxide, chloroform, trichloroethylene or diethyl ether. Ostergren's findings offered one explanation for the previously observed, anesthetic induced inhibition of cell division. Interest in this topic was revived when in 1956, Lassen et al. [91], reported that severe bone-marrow depression leading to granulocytopenia and t h r o m b o c y t o p e n i a , occurred in tetanus patients who received prolonged nitrous oxide anesthesia. Because all patients studied also received other drugs, there was still d o u b t concerning the relationship between nitrous oxide administration and bone-marrow depression [54]. Nonetheless, the report stimulated numerous researchers to examine effects of anesthetics on the dividing cell. 10 years after Lassen's paper, Andersen [3] reviewed the effects of central nervous system depressants on mitosis. Included in his review were the inhalational anesthetics, diethyl ether, chloroform, nitrous oxide, cyclopropane and ethylene. Andersen concluded that these anesthetics inhibited mitosis by an effect on the mitotic spindle. Furthermore, inhibition usually occurred at concentrations within the clinical range and was compatible with the effect of colchicine and chloral hydrate which are the most specific and effective c-mitotic agents. Several reports published after Andersen's review indicated that interference with the normal mitotic phase of cell cycle may lead to nuclear abnormalities. Sturrock and Nunn [122] using Chinese hamster fibroblasts, showed that the rate of formation of binucleate and trinucleate cells and cells with micronuclei increased following halothane exposure. They also demonstrated a synergism between halothane and nitrous oxide in the production of nuclear abnormalities [122]. Kusyk and Hsu [89] showed that halothane, enflurane and methoxyflurane induced segregational errors of chromosomes in mammalian cells grown in culture and in avian cells exposed in vivo. In a series of articles, Grant et al. [63--66] reported that inhalational anesthetics induced chromosomal abnormalities in the broad bean (Vicia faba) root tip cells. Finally, a recent study b y Coate et al. [30], demonstrated that exposure to t w o concentrations of a combination of nitrous oxide and halothane increased the number of chromosomal aberrations in rat spermatogonial cells. Such studies indicate that anesthetics have potential for causing chromosomal mutations. In addition to effects on mitosis, it is now clear that anesthetics inhibit other phases of the cell cycle. For example, Bruce and Traurig [24] examined the effects in vivo of 0.1 or 0.5% halothane on the cell cycle. In later studies, Sturrock and Nunn [121,123] examined the effects of halothane on the G1 and S

179 phases of Chinese hamster fibroblasts grown in culture. They showed that halothane produced a slight b u t significant dose related depression of [3H]thymidine uptake even at clinically used concentrations. In contrast to the results obtained by Bruce and Traurig, they also showed that there was delayed onset of S phase, indicating a prolongation of G1 phase. Numerous other cellular effects of anesthetics have been noted. They include effects on cell metabolism, movement, excitable and nonexitable lipid membranes, receptors and other proteins. Ethylene is particularly n o t e w o r t h y n o t only as an anesthetic but also as a plant hormone in concentrations as little as 0.005 p p m [5,16,25,26,49,50,80--82,90]. However, other anesthetics do n o t appear to have plant-hormone activity [113]. In the present review, the authors have chosen to present only a few salient references to highlight the widespread effects that anesthetics have on cells. No a t t e m p t has been made to review comprehensively this extensive topic. For a more complete treatment, the interested reader is referred to three detailed publications [54,55,78].

Mutagenicity Trichloroethylene Of all the anesthetics, trichloroethylene (TCE) has been the most studied. It was the first halogenated agent to gain widespread clinical acceptance and is also produced in large quantities for numerous other commercial uses. TCE is an improtant industrial pollutant and has been found in increasing concentrations throughout many parts of the world [57]. Greim et al. [67,68] reported that TCE was mutagenic in E. coli K12 strain. Bacteria were suspended in an incubation mixture containing microsomal proteins and cofactors and various concentrations of TCE. After a 2-h incubation period at 37°C the cells were plated on selective media to determine the number of mutant colonies and on nonselective media to determine survival. At 3.3 mM TCE, survival was 76% and these was an increase of approx. 2.3-fold in the number of arg* revertants. Under the test conditions, there were no increases in the back mutation systems nad÷ or gal ÷ nor increase of forward mutation to 5-methyltryptophan resistance. In contrast to these findings, Uehleke et al. [128] reported that TCE was n o t mutagenic in E. coli K12. The Ames Salmonella/mammalian microsome system [2] has been used by a number of inveetigators to examine the mutagenicity of TCE. Cern~ and Kyp~nov~ [28] reported that TCE was mutagenic to S. typhimurium strains T A 1 5 3 5 and T A 1 5 3 8 in vitro w i t h o u t metabolic activation. They also showed that TCE increased the number o f revertants of strain TA1950, TA1951 and T A 1 9 5 2 in vivo in a host-mediated assay using female ICR mice. Dose-dependence was demonstrated only when TCE was tested in vitro. Simmon et al. [116], and Baden et al. [12], reported that TCE at vapor concentrations above 1% was mutagenic in assays with S. typhimurium strain TA100. These assays were c o n d u c t e d b y testing TCE in sealed desiccators. Metabolic activation was provided by $9 liver homogenate preparations from male Fischer 344 rats or male B6C3/FI mice which had been induced with a single intraperitoneal injection of Aroclor 1254 (500 mg/kg). The increase in

180 revertants was reproducible and dose-related, but less than 2-fold above control. Mutagenic activity was not observed in the absence of the liveractivation systems nor with strain TA1535. In addition, experiments conducted with reaction mixtures in liquid suspension gave negative results [12]. The T C E samples used in these studies were free of epichlorohydrin and 1,2-epoxybutane as determined by GC/MS. Furthermore, had these contaminants been present, mutagenicity should have been observed in the absence of the liver homogenate systems. Trace concentrations of these contaminants are suspected of contributing to the carcinogenic activity of T C E in animal bioassays [74, 137]. Other studies using the Salmonella system have been negative. Uehleke et al. [129], reported that neither S. typhimurium strain TA1535 n o t TA1538 was reverted to histidine independence by 3.6 mM TCE in the presence of metabolic activation. They did find, however, that after an intraperitoneal injection of 14C-labeled TCE in mice, metabolites were b o u n d preferentially to liver endoplasmic reticulum and lipid [128]. Bartsch et al. [17] and Barbin et al. [15], reported that TCE vapor in air was n o t mutagenic to strain TA100 when tested in the presence of $9 prepared from phenobarbital pretreated mice. However, few details of the exact m e t h o d o l o g y were described. Henschler et al. [72], also reported that TCE was n o t mutagenic to strain TA100 when tested in the presence of PCB (Aroclor 1254) induced rat-liver microsomes using the standard agar-overlay procedure. It is probable, however, that because of its extreme volatility, TCE did n o t remain for long in the presence of bacteria or microsomes. For this reason, the agar-overlay procedure is an extremely insensitive m e t h o d for the detection of volatile mutagens. In the same paper, the investigators confirmed that the contaminants of TCE, epichlorohydrin and 1,2-epoxybutane, were highly mutagenic [72]. In several other papers, Henschler et ai. [70,71,73], discussed the metabolism and structural activity relationship of chlorinated ethylenes. Infante [77] has also discussed the mutagenic risks associated with TCE. Finally, Waskell [137] exposed S. typhimurium strains TA98 and TA100 to 0.5--10% vapor concentrations TCE in sealed vials for 48 h, and observed no increase in revertants either in the presence or absence o f liver homogenate prepared from rats. The sex and strain of the rats or use of an enzyme inducer prior to the preparation of the metabolic system were not indicated. On the other hand, chloral hydrate, a presumed metabolite of trichloroethylene [137], was weakly mutagenic in strain TA100, b u t n o t TA1535 or TA98. Metabolic activation (liver homogenate) was required. Waskell [137] also found that 50 pl TCE did n o t increase the n u m b e r of single-strand DNA breaks when incubated under physiologic conditions with a 0.4-ml reaction mixture containing doublestranded circular radioactive phage PM2 DNA. A number of TCE mutagenicity studies have used yeast as the test organism. Shahin and yon Borstel [115] observed increases in reversion in Saccharomyces cerevisiae strain XV185-14C with several concentrations (0.1--20 #l/ml cells) of TCE in the presence of a metabolic activation system prepared from male mouse liver. Reversion at 3 loci, lysl-1, his1-7 and horn3-10, was reported at survivals of less than 1%. However, even at those low survivals, there were increases from 2- to 6-fold in the number of revertant colonies. There was no

181 mutagenic activity in the absence of the metabolic system. The authors concluded that TCE was a powerful mutagen and induced ~frameshift as well as base substitution mutations. TCE (0.2 ml/6.2 ml reaction mixture) induced reversion (point mutation) at the ilv locus (3.8 fold m a x i m u m ) and mitotic gene conversion at the trp locus (2.5-fold m a x i m u m ) in S. cerevisiae D7 when assayed in suspension for 4 h at 37°C with a metabolic activation system prepared from livers of male CD-1 mice [20]. The increases at both loci were reproducible and dose-related. They were n o t seen w i t h o u t metabolic activation. In intra-sanguinous host-mediated assays using mice as the host, a single oral dose of 400 mg/kg TCE increased gene conversion in S. cerevisiae D4 at the trp and ade loci. The increases were greatest in cells recovered from the liver (1.8- to 2.6-fold) and kidney (2.0- to 2.5-fold) and least from cells recovered from the lungs. When TCE was administered subacutely to the mice (150 mg]kg]day, 5 days a week, 22 administrations plus 400 mg/kg on the day the yeast were assayed), increases in convertants of 3- to 10-fold above control were obtained in cells recovered from the kidney and liver. A single oral dose of TCE 400 mg/kg also increased gene conversion (trp locus) and reversion (ilv locus) when S. cerevisiae D7 was tested in the intrasanguinous host-mediated assay. Increased convertants were recoverd from the kidney and liver, but n o t lungs. The increases in trp convertants were greater with D7 than with D4, suggesting that strain D7 may be a more sensitive strain to the mutagenic effects of the TCE metabolite. Unfortunately, no comparison of the relative sensitivity of these two indicator strains was made in vitro. A n o t h e r mutagenicity assay which has been used to examine TCE is the mammalian spot test in mice. Fahrig [52,53] showed t h a t an intraperitoneal injection of 1 mmole/kg TCE resulted in an increased number of colored spots on the adult mouse black coat indicating an increase in either gene mutations or recombinational processes. When taken together, the above studies provide good evidence that TCE has weak to moderate genetic activity. If one only included studies using uncontaminated TCE, one would still conclude that TCE was genotoxic; however, the evidence would be weakened considerably.

Fluroxene Baden et al. [8,10] showed that the volatile anesthetic fluroxene was mutagenic to S. typhimurium strains TA1535 and TA100 and E. coli strain WP2 when assayed in liquid suspension but n o t when assayed in desiccators. A mutagenic response occurred at vapor concentrations between 3 and 30% only in the presence of enzymes prepared from livers of Aroclor 1254 pretreated rodents. Mutagenic activity was not seen with S. typhimurium strains TA1537 and TA98 or with enzymes from humans or uninduced rodents. Trifluoroethanol, a metabolic of fluroxene, and the urine from rats anesthetized with fluroxene were n o t mutagenic. The authors concluded that fluroxene was a weak promutagen which required preincubation with liver enzymes before it is mutagenic. In the only other study with fluoxene, White et al. [140] showed t h a t it increased sister-chromatid exchanges (SCE) in Chinese hamster ovary cells. Exponentially dividing cells were exposed for 1 h to a 2.47% vapor con-

182 centration (1 MAC)*; SCE were increased significantly from 0.536 + 0.018 to 1.147 + 0.030 per chromosome.

Divinyl ether Divinyl ether has been reported by Baden et al. [12] to be mutagenic to strains TA100 and TA1535 in desiccator and liquid incubation when tested in the Salmonella/mammalian microsome system. In desiccators, a dose-dependent increase in revertants occurred after an 8-h exposure to divinyl ether in the absence of metabolic system ($9 mix) above 1% vapor concentrations. The mutagenic response was enhanced (6-fold maximum) when $9 mix was added. After a 2-h liquid incubation, a mutagenic response occurred at vapor concentrations between 1 and 30% only in the presence of the $9 mix. As with fluroxene, the genotoxic effect of divinyl ether was observed in the SCE system. White et al. [140], reported that 1-h exposure to 1.79% divinyl ether (1 MAC) significantly increased the rate of SCE in Chinese hamster ovary cells from 0.536 + 0.018 to 0.706 + 0.022 per chromosome. It is of interest that both fluroxene and divinyl ether are mutagenic at clinically used concentrations. Other anesthetics Nitrous oxide, cyclopropane, diethyl ether, halothane, chloroform, enflurane, isoflurane and methoxyflurane were n o t mutagenic when tested in the Salmonella/mammalian microsome system using a variety of bacterial strains and test conditions and at multiple anesthetic concentrations [7,9,12,67,116, 128,129,137]. Furthermore, the urine of patients anesthetized with halothane, enflurane, isoflurane, or methoxyflurane showed no mutagenic activity when assayed directly in standard agar-overlay studies [7,9]. However, no attempt was made to test urine which had been concentrated, a technique which would have greatly increased the sensitivity of the assay. In addition to the anesthetics, several of their metabolites have been tested in the Salmonella system. Trifluoroacetic acid, trifluoroethanol and trifloroacetaldehyde hydrate were n o t mutagenic when tested at various concentrations in the standard agar-overlay assay [7,137]. A presumed halothane metabolite, 1,1-difluoro-2-bromo-chloroethylene (CF2CBrC1) was n o t mutagenic in the standard Salmonella assay [48, 138] b u t was weakly mutagenic when tested with exponentially growing bacteria [48]. Of t w o volatile halothane metabolites tested, 1,1,1-trifluoro-2-chloroethane (CF3CH2C1) was n o t mutagenic [48,138] whereas 1,1-difluoro-2-chloroethylene (CF2CHC1) was weakly mutagenic [48,61]. Finally, urine of anesthesiologists has been examined for mutagenic activity using the Salmonella system. An early report indicated that 11 o u t of 15 anesthesiologists working in unscavenged operating rooms, had mutagens in their urine [98]. However, a later study with a larger number of operating r o o m personnel, did n o t confirm the earlier findings [ 11 ]. In addition to assays with Salmonella, halothane and chloroform have been tested with E. coil strain K12 [85,128] and diethyl ether has been tested with a DNA polymerase-deficient m u t a n t of E. coil, P3478 [58]; none was mutagenic. * M A C is t h e m i n i m a l a l v e o l a r c o n c e n t r a t i o n r e q u i r e d t o p r e v e n t r e f l e x a c t i v i t y i n 50% o f p a t i e n t s s u b j e c t e d to a n o x i o u s s t i m u l u s .

183 With the exception of cyclopropane, all the above agents have been tested at a 1 MAC concentrations for 1 h using the Chinese hamster ovary cell SCE assay, again with negative genotoxic results [140]. Nitrous oxide, halothane, chloroform and enflurane have also been tested for mutagenic effects using resistance to 8-azaguanine in Chinese hamster lung fibroblast cells grown in culture [119,120]. Cells were exposed to 75% nitrous oxide, 1--3% halothane, 1--3% chloroform or 1.5--6.5% enflurane for 24 h. No significant increase in numbers of mutations was found. However, no metabolizing system was added to the test system. It is of interest that although chloroform and nitrous oxide were n o t mutagenic when assayed alone in these systems, both agents have been found to potentiate the action of ionizing radiation [4,42,59,111]. For example, A n t o k u [4] showed that a saturated solution of nitrous oxide increased the yield of DNA-strand breaks in mouse leukemic L5178Y cells exposed to 6°Co ~-rays. Such studies demonstrate that anesthetics may interact with other chemical or physical factors to produce mutagenic effects. Several investigators have used Drosophila to examine the mutagenic potential of anesthetics. In an early study, Morgan [102] showed that diethyl ether delivered by inhalation daily for a b o u t 11 days, did n o t produce mutations in Drosophila ampelophila. In another report, Krechkovsky and Shkvat [87] noted that halothane had no mutagenic effect on mature sex cells of male Drosophila melanogaster. These authors exposed flies to halothane for 10 min, 3--4 times a day, for 12 days. On the other hand, Garrett and Fuerst [60] demonstrated that nitrous oxide increased the number of recessive sex-linked lethal mutations in Drosophila melanogaster. Flies gassed in a 100-ml flask for 1 min to 22 ml/min nitrous oxide, had a lethal mutation rate of 2.82 + 0.03, a value significantly higher than air-treated controls. The only report containing positive results with a m o d e m volatile anesthetic is that by Kramers and Burm [86]. There investigators performed mutagenicity studies with halothane in Drosophila melanogaster using sex-linked recessive lethals as an indicator of genetic damage. Adult males were exposed to halothane either for 14 days at 1000 or 1600 p p m (v/v) or for 1 or 2 days at 2100 or 20 000 ppm. Results for experiments at the high vapor concentrations were uniformly negative. In several of the low concentrations, long-term experiments, a slight increase in mutation frequency was observed. Pooled data from these latter studies just reached significance at a 5% level. The authors concluded that their results were of borderline significance b u t indicated with a fair degree of probability that halothane was weakly mutagenic under the conditions used. It is clear that further studies with halothane, nitrous oxide and other anesthetics using the same model system should be performed to assess the significance of these Drosophila experiments. Finally, using t w o clones (02 and 4430) of Tradescantia, Sparrow and Schairer [117] showed that nitrous oxide increased the mutation rate of cells in petals and stamen hairs. However, the maximum response rate was little more than twice the spontaneous rate and the authors concluded that nitrous oxide was at best a weak mutagen.

184 TABLE 3 MUTAGENICITY

TESTS

Anesthetic

Positive

Negative

Halothane

D D, ------A, A, A,

A, A, A, A, A, A, A D, A, ---

Nitrous oxide Chloroform Enflttrane

Methoxyflurane Iso flttrane

Cyclopropane Diethyl ether Trichloroethylene Fluroxene Divinyl ether

T

M, S SCE SCE

8-AzG, 8-AzG, 8-AzG, 8-AzG, SCE SCE

D, S C E SCE SCE SCE

SCE SCE

A, A m e s SalmoneLla o r E s e h e r i c h i a f l n a m m a l i a n m i c r o s o m e . 8 - A z G , 8 - a z a g u a n i n e , C h i n e s e hamster lung f i b r o b l a s t s . D~ D r o s o p h i l a . M, M o u s e s p o t t e s t . S, S a c c h a r o m y c e s . S C E , S i s t e r - c h r o m a t i d e x c h a n g e , C h i n e s e h a m s t e r ovary cells. T, Tradescantia.

Effects o f nitrous oxide on plants In 1954, Ostergren [107] reported that when plants of Crepis capillaris were treated with 10 atmospheres pressure nitrous oxide for 4--6 h at the time when the first or second zygotic divisions were occurring in their pollinated flowers, a fair yield of polyploid and aneuploid plants was obtained in their offspring. Subsequently, the same author using a similar m e t h o d of exposure, demonstrated the same effect in Phalaris canariensis and Ph. paradoxa [108]. Since the pioneering w o r k of Ostergren, numerous investigators have shown that various species of plants exposed to high pressure nitrous oxide produce numerous aneuploid or polyploid progeny [41,45--47,100,101,105,124--126]. Although the efficiency of this technique varies in different plant species, it has occasionally proved a viable alternative to treatment with colchicine, a drug classically used in higher plants to produce chromosome doubling. In addition, exposure to high pressures of nitrous oxide has been a valuable genetic tool for elucidating the mechanisms and timing of chromosome movement [19]. In the strict sense, aneuploidy and polyploidy are heritable changes in genetic material and therefore fall in the category of mutagenicity. However, these effects have only been demonstrated in plant species and only at high pressures. It is unlikely that such effects would occur in humans exposed to low concentrations of nitrous oxide at atmospheric pressure. Summary and conclusion Because of positive results obtained with nitrous oxide or halothane in the Drosophila or Tradescantia assays, and the weakly positive results with some volatile halothane metabolites in the Salmonella assay, one cannot be dogmatic concerning the lack of mutagenicity of these anesthetics. Nonetheless, the Salmonella/mammalian microsome, the SCE and the 8-azaguanine test systems are

185 n o w considered well-validated methods for the detection of chemical mutagens/carcinogens and have been applied fairly systematically ti inhalational anesthetics. A summary of results obtained from studies which have used these assays and other test systems is shown in table 3. Taken together, they provide reasonable evidence that under normal conditions m o d e m inhalational anesthetics and many previously used anesthetics are not mutagens and therefore, probably not carcinogens. On the other hand, the anesthetics which contain the vinyl moiety are mutagens and should be considered potential carcinogens. References I Ad hoc committee on the effect of trace anesthetics on the health of operating r o o m personnel, A m e r i c a n S o c i e t y o f Anesthesiologist, O c c u p a t i o n a l Disease" A m o n g O p e r a t i n g R o o m P e r s o n n a l : A N a t i o n a l S t u d y , A n e s t h e s i o l o g y , 41 ( 1 9 7 4 ) 3 2 1 - - 3 4 0 . 2 Ames, B.N., F.D. Lee a n d W.E. 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