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Q) Leukemia (2001) 15, 10-20 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu EDITORIAL Hypothesis: Phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia TA McDonald1, NT Holland2, C Skibola2, P Duramad2 and MT Smith2 10ffice of Environmental Health Hazard Assessment, California Environmental Protection Agency, Oakland; and 2Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, California, USA High background levels of phenol and hydroquinone are present in the blood and urine of virtually all individuals, but vary widely. Phenol and hydroquinone have been strongly implicated in producing leukemia associated with benzene exposure, because they reproduce the hematotoxicity of ben zene, cause DNA and chromosomal damage found in leukemia, inhibit topoisomerase II, and alter hematopoiesis and clonal selection. The widely varying background levels of phenol and hydroquinone in control individuals stem mainly from direct dietary ingestion, catabolism of tyrosine and other substrates by gut bacteria, ingestion of arbutin-containing foods, cigarette smoking, and the use of some over-the-counter medicines. We hypothesize that these background sources of phenol and hydroquinone and associated adducts play a causal role in pro ducing some forms of de novo leukemia in the general popu lation. This hypothesis is consistent with recent epidemiolog ical findings associating leukemia with diets rich in meat and protein, the use of antibiotics (which change gastrointestinal flora make-up), lack of breastfeeding, and low activity of NAD(P)H quinone oxidoreductase which detoxifies quinones derived from phenol and hydroquinone and protects against benzene hematotoxicity. An attractive feature of our hypothesis is that it may explain why many people who have no known occupational exposures or significant smoking history develop leukemia. The hypothesis predicts that susceptibility to the dis ease would be related to diet, medicinal intake, genetics and gut-flora composition. The latter two of these are largely beyond our control, and thus dietary modification and reduced use of medicines that elevate phenol levels may be the best intervention strategies for lowering leukemia risk. Leukemia (2001) 15, 10-20. Keywords: phenol; hydroquinone; diet; gastrointestinal flora; leuke mia; benzene Introduction The lifetime risk of dying from leukemia in the US is approxi mately eight in 1000, or close to 1%, and approximately 30 000 new cases of leukemia are diagnosed every year.1 The causes of the vast majority of these leukemia cases in the gen eral population are unknown.2 Numerous studies have exam ined possible links to genetics, infectious agents, occupational and environmental exposures to radiation, smoking, pesticides and solvents, and chemotherapy. However, very few have investigated possible dietary links with leukemia, despite the fact that diet is one of the most important causes of cancer.3 In this paper, we propose the hypothesis that phenol and Correspondence: MT Smith, School of Public Health, Division of Environmental Health Sciences, 140 Warren Hall, University of Cali fornia, Berkeley, California 94720-7360, USA; Fax: 510 642-0427 Disclaimer: The views expressed by Dr McDonald do not necessarily represent those of the Office of Environmental Health Hazard Assess ment, the California Environmental Protection Agency or the State of California. Received 22 May 2000; accepted 12 September 2000 hydroquinone (1,4-dihydroxybenzene), derived mainly from dietary sources, are significant contributing factors in producing de novo leukemia in the general population. This hypothesis stems from several lines of research that have converged in recent years. First, over the past few dec ades scientists have conducted intensive investigations to understand the mechanism(s) by which benzene causes leuke mia. Although the precise mechanism is not known, evidence continues to mount toward one prevailing theory in which the metabolites, phenol and hydroquinone, are central players.4,5 Second, investigators involved in developing biomarkers of exposure or in understanding the pharmacokinetics of ben zene, medicinal products and other chemicals in humans have observed high and widely varying background levels of phenol, hydroquinone and other phenolic species in the blood and urine of control individuals (see below). Third, recent studies on genetic susceptibility suggest that quinones and oxidative stress are key risk factors for leukemia.6-9 Finally, recent epidemiological studies have identified that consumption of high meat diets,10,11 use of antibiotics,12 and absence of breastfeeding13-16 are risk factors for leukemia. A hypothesis that reconciles all of these different lines of research is that phenolic species derived mainly from dietary sources are involved in generating de novo leukemia in the general population and that exposure to benzene and other environmental leukemogens adds to those rates. In this paper, we review the evidence that implicates phenol and hydroquinone as risk factors in leukemia, present obser vations of high background levels of these compounds in human populations, describe the many sources of these phe nolic compounds, and discuss additional factors that affect the variability in background levels. A discussion of the predictions and criticisms of the hypothesis follows. Evidence suggesting that phenol and hydroquinone are causal factors in leukemia Phenol and hydroquinone play an important role in benzene-induced leukemia Benzene and ionizing radiation are among the few established environmental causes of leukemia in humans. Although the precise mechanism by which benzene induces leukemia is not known, the prevailing hypothesis is as follows (Figure 1).4,5 Benzene is metabolized in the liver primarily by cytochrome P4502E1 (CYP2E1), first to benzene oxide and then to phenol, hydroquinone and other polyphenolic metabolites. These phenolic metabolites can be detoxified by conjugation with sulfate, glutathione or glucuronide. Sulfation may not be a potent detoxification mechanism, because the bone marrow contains high levels of sulfatase that can break down the sulfur Editorial TA McDonald et al 11 Figure 1 Benzene metabolism and primary carcinogenic pathways. P450, cytochrome P450; MPO, myeloperoxidase; NQO1, NAD(P)H: quinone oxidoreductase. conjugates to free phenols.17 The phenolic metabolites also travel to the marrow where they are converted to highly reactive quinones by peroxidases, such as myeloperoxidase, or through autooxidation.4,5 The major defenses against these toxic quinone products include reduction via NAD(P)H: quin one oxidoreductase (NQO1) or conjugation with gluta thione.18,19 Quinone oxidation products form DNA adducts and induce a wide-range of DNA damage both directly and indirectly (see below). These quinone metabolites also increase oxidative stress20 and alter differentiation and cell growth in the myeloid compartment.21-23 This combination of genetic and epigenetic effects on bone marrow progenitor cells leads to the production of leukemia in some exposed individuals. When phenol and hydroquinone are administered together, they reproduce benzene's myelotoxic effects.24,25 Phenol and phenol-derived metabolites (eg 4,4'-diphenoquinone) inhibit topoisomerase II and enhance the genotoxic effects of hydro quinone.26 Many chemotherapeutic drugs that inhibit topoiso merase II also induce leukemias.27 The co-administration of phenol and hydroquinone to mice induces micronuclei and oxidative DNA damage in the bone marrow in a manner simi lar to benzene.26,28,29 Further, combinations of the phenolic metabolites of benzene enhance DNA adduct formation.30,31 It has therefore been suggested that all the phenolic metab olites of benzene play a role in benzene-induced toxicity, and that it is the combination that is most toxic.24,32 Phenol and hydroquinone produce genetic damage of the type found in leukemia Quinones derived from phenol, catechol, hydroquinone and 1,2,4-benzenetriol cause various forms of genetic damage including chromosome breakage and aneuploidy.26,33 Aneuploidy is the loss or gain of whole chromosomes and is a fre quent clonal aberration in leukemia. Common aneuploidies include trisomy of chromosome 8 and monosomy of chromo somes 5 and 7.34,35 The phenolic metabolites of benzene have been shown to induce trisomy 8 and monosomy 5 and 7 in cultured human cells in vitro, including CD34+ progenitor cells.36-38 Workers exposed to benzene also have higher lev els of these aneuploidies in their peripheral blood.39 Through their inhibitory action on topoisomerase II, binding to other DNA-associated proteins and formation of DNA adducts, phe nol and hydroquinone cause double-strand breaks and chro mosome breakage.40,41 This has been shown in vitro to lead to deletions and translocations commonly found in leukemia, including del(5q) and del(7q).38 Again, workers exposed to benzene have been shown to have higher levels of del(5q), del(7q) and translocation (8;21) in their blood cells.39,42 Mice treated with phenol and hydroquinone also show elevated levels of aneuploidy and chromosome breakage in their bone marrow.26 Hydroquinone produces epigenetic changes that increase the risk of developing leukemia Other research has focused on the possible epigenetic mech anisms involved in benzene-induced leukemia. Hydro quinone has been shown to alter hematopoietic stem cell pro liferation and differentiation in the myeloid compartment.21,22 Related effects may include alteration of apoptosis and alter ation of clonal expansion of blood progenitor cells (reviewed in Ref. 43). These perturbations may proceed through a variety of means including alteration of inflammatory mediators, growth factors and other cellular messengers.44,45 Irons and Leukemia Q) Editorial TA McDonald et al 12 coworkers' demonstrated that treatment of mouse of human Phenol and hydroquinone levels in unexposed individuals marrow progenitor cells of the granulocyte-macrophage lin eage with hydroquinone increased the number of colonies High background concentrations of phenol, hydroquinone, dependent on granulocyte-macrophage colony-stimulating- catechol and 1,2,4-benzenetriol have been measured in the factor (reviewed in Ref. 21). In theory, this increase provides blood, urine and intestines of presumably unexposed more targets for the genotoxic effects of phenol, hydroquinone humans48-62 and in the blood and urine of rodents.63-65 Rep and related species.4 resenting measurements of hundreds of individuals in total, these studies showed that mean urinary phenol and hydro quinone levels were about 10 p.p.m. (13 pg/mg creatinine) and 4 p.p.m. (5 pg/mg creatinine), respectively. The urinary NQO1 detoxifies quinones derived from phenol and hydroquinone and protects against leukemia and benzene-induced hematotoxicity concentrations of phenol and hydroquinone varied widely (ie 5- to 25-fold) within a given population.49-51 Some studies have reported differences in phenol levels between sexes,54 whereas others have not.48 Variability of phenol levels among individuals appears to be greater than the difference among Additional evidence that the phenolic metabolites of benzene males and females.48 Urinary concentrations of phenol have play a central role in benzene-induced leukemogenesis comes been observed to be approximately 10-fold higher than con from investigations of key enzymes involved in the activation centrations in serum from the same individuals,59 which may or detoxification of quinones (Figure 1). These enzymes reflect the rapid removal of phenol from the blood and include CYP2E1, which converts benzene to phenol and phe excretion via urine. Based on mean 24-h urinary levels in con nol to hydroquinone, and NQO1, which reduces benzoqui- trol individuals49,50 and assuming a daily fluid intake of 2 lit none back to hydroquinone and lowers oxidative stress.5 In ers, we estimate that humans produce endogenously or ingest benzene-exposed workers, a high activity of CYP2E1 and each day roughly 0.2 mg/kg of phenol, 0.1 mg/kg hydro homozygous inheritance of a mutation in the NQO1 gene that quinone, and 0.3 mg/kg of catechol, with considerable inter eliminates NQO1 activity conferred a 7.6-fold increase in risk individual variability expected. of hematotoxicity.6 Interestingly, inheritance of the same inac Interestingly, high background levels of covalent macromo- tivating C609T mutation in the NQO1 gene also confers lecular adducts of benzoquinone (the reactive metabolite of increased risk of therapy-related leukemia,7 de novo leuke hydroquinone) were observed in the blood of humans66,67 and mia9 and infant leukemia with MLL gene rearrangements.8 in the blood and bone marrow of rodents.67-69 The adduct Recent work from our laboratory suggested that NQO1 pro levels were equivalent to those resulting from a relatively high tects the marrow against hydroquinone- and 1,2,4-ben- exposure to benzene.67,68 Several investigators have estimated zenetriol-induced toxicity through an unexpected mechanism, that exposures to benzene far in excess of the current work inhibition of high molecular weight DNA adducts.31 place standard would be required to significantly increase the adduct levels over background levels.65,67,68 These obser vations prompted us to speculate on the pathological conse quences that background levels of phenol and hydroquinone Phenol and hydroquinone levels in benzene-exposed individuals may have for the general population. A number of studies have also assessed phenol levels in patients with different diseases, including colon cancer, fam ilial polyposis, and Crohn's disease.54 Patients with diverticu Historically, when occupational exposures to benzene were lar disease or polyposis with ileorectal anastamosis appeared high (eg >50 p.p.m.), urinary phenol levels were seen as a to have elevated urinary phenol levels (about 2-fold) relative good biomarker for exposure to benzene.46-48 Studies, such to normal individuals. The most noticeable increase in phenol as those conducted by Inoue et al,49,50 observed a significant levels was observed among patients with Crohn's disease, correlation between air concentration of benzene in breathing which is a common type of inflammatory bowel disease zone and phenol or hydroquinone concentrations in urine (Figure 2). Phenol levels among Crohn's patients were (corrected for creatinine and specific gravity) (Figure 2). The approximately 4- to 30-fold higher than levels among normal authors reported a significant difference between urinary phe subjects. Interestingly, in agreement with our hypothesis, there nol levels in a group exposed to 10 p.p.m. benzene compared are studies suggesting that Crohn's patients have an increased to unexposed workers. Similarly, a dose-related increase in risk of leukemia.70 urinary phenol levels was observed among groups of Chinese Thus, based on the strong evidence implicating phenol and workers exposed to benzene51 (Figure 2). Workers exposed to hydroquinone in benzene-induced leukemia coupled with the 0, 1 to 31 p.p.m., or >31 p.p.m. exhibited urinary phenol observations of high background levels of these compounds concentrations of 4 to 55 p.p.m., 16 to 487 p.p.m., or 28 to in the general population, we hypothesized that these back 517 p.p.m., respectively. These findings indicate that high ground concentrations and associated adducts may play a benzene exposure significantly affects the levels of excreted causal role in de novo leukemia. phenol (Figure 2a). However, in these and other studies, uri nary phenol and hydroquinone were found to be poor predic tors of lower benzene exposures.48-52 For example, urinary Sources of phenol and hydroquinone in 'unexposed' people phenol levels of workers exposed to 1 to 5 p.p.m. benzene were indistinguishable from levels in unexposed individuals.50 Our investigation of the available literature has uncovered a Likewise, the distributions of urinary hydroquinone levels of surprising number of potential sources of the background levels unexposed individuals overlapped with urinary levels from of phenol and hydroquinone. These include over-the-counter female workers exposed to benzene between 9 and 14 p.p.m. medicines, smoking, numerous foods and beverages, and the (7 h, time-weighted average).49 catabolism of protein (tyrosine) and other substrates by the gut Leukemia Editorial TA McDonald et al 13 Figure2 Levels of phenol (a) and hydroquinone (b) in the urine of various groups of individuals. Phenol and hydroquinone levels in the blood and urine are commonly analyzed by gas chromatography in combination with various methods of initial purification and hydrolysis of the specimen. In the earlier studies colorimetric methods of Gibbs or Theis-Benedict were used to quantitate phenol (reviewed in Ref. 48), which were less specific and sensitive compared to gas chromatography. The other important factors affecting reported phenol values are variation in sampling and adjustment for gravity and creatinine. Phenol levels are typically reported as a concentration, mass per mass creatinine, or total mass excreted in 24 h. Data from Refs 47, 50, 51, 54, 57, 58, 75, 107, 108. flora. Below we discuss these sources and also describe additional host and environmental factors that affect the varia bility of the background levels of the phenolic compounds. Medicines Phenol is used in some relatively unusual medications and treatments, which result in sharp but transitory increases in blood phenol levels.71-74 Of more importance to the general population is the use of some over-the-counter medications such as Pepto-Bismol and Chloraseptic lozenges.75 In a trial of Pepto-Bismol, urinary phenol concentration increased over 40-fold, reaching a concentration of 260 p.p.m. in a volunteer who ingested 1 oz of Pepto-Bismol every hour for 8 h (Figure 2). Additional experiments indicated that phenyl sal icylate in Pepto-Bismol was responsible for the increase in urinary phenol levels. Fishbeck et a/75 also observed increases in phenol levels in human subjects after ingestion of Chlora septic lozenges. The total urinary phenol rose to a maximum of 270 p.p.m. These studies show that high urinary phenol levels can be achieved without any significant exposure to benzene. Smoke Cigarette and wood smoke contain phenol, catechol, hydro quinone as well as benzene.76-78 Amounts received from nonfiltered mainstream cigarette smoke were estimated to be 60- Leukemia Q) Editorial TA McDonald et al 14 140 phenol per cigarette, 140-500 ^g catechol per ciga Production of pheno/ and other pheno/ics by the gut rette, and approximately 150-430 ^g hydroquinone per ciga flora rette.77 Haufroid et a/79 observed statistically significant dose- related increases in the urinary concentrations of phenol, hydroquinone and catechol associated with the number of cigarettes smoked. Cigarette smoking is associated with an approximately 50% increase in leukemia risk.80,81 Gut microflora may play an important role in the biotransformation of chemical and dietary components into procarcinogenic compounds in the gut. Smith and Macfarlane62 have shown that simple phenols are major products of tyrosine metabolism by gut microflora in the distal colon. Tyrosine is an aromatic amino acid that may be ingested Diet through the diet or formed from the essential amino acid, phe nylalanine, through an irreversible reaction catalyzed by phe nylalanine hydroxylase. Tyrosine and phenylalanine are found Although the use of cigarettes and certain medicines can result in high concentrations in milk, beef and eggs. Phenylalanine in significantly elevated levels of phenol and hydroquinone in is also commonly used as an artificial sweetener in a number some groups of individuals, diet is a significant source con of sugarless foods and beverages. Similarly, some commonly tributing to increased tissue levels of phenol and hydro occurring plant phenolics are efficiently metabolized by the quinone for all persons. Phenol and hydroquinone are derived gut flora of rodents to phenol, catechol and other simple phe from the diet both directly and indirectly. Many common nols.82,83 Escherichia co/i can convert glucose to catechol foods and beverages contain phenol and hydroquinone. A through a minor metabolic pathway;84 however, the amount major source of phenol stems from catabolism of protein and of catechol that might be generated by gut flora from sugars other compounds by gut bacteria, which appears to depend is unknown. highly on the metabolic activity of the intestinal bacterial Endogenous bacterial strains that convert tyrosine to phenol microflora.57,62 A potentially significant source of hydro- include the obligate anaerobes, Bacteroides fragi/is, Pepto- quinone comes from ingestion of foods containing arbutin, a streptococcus asaccharolyticus, and the facultative anaerobes, naturally occurring plant product that is converted to hydro- Escherichia co/i, Proteus sp., Staphy/ococcus faeca/is and Sta- quinone by stomach acids. The wide inter-individual varia phy/ococcus a/bus (Figure 3).54,62,85 Studies of human colonic bility in urinary levels of phenol noted above may reflect the contents in sudden death victims in the UK showed that wide range of direct intake of phenolic-containing foods anaerobic bacteria outnumber facultative microorganisms in and differences among individuals in the composition and the human gut by two to three orders of magnitude with Bac chemistry of the gut flora. teroides fragi/is being the most numerous86 (Figure 3). How Phenol, hydroquinone, catechol, and 1,2,4-benzenetriol ever, the composition of intestinal bacteria varies considerably are found in a wide variety of foods and beverages. As shown among different populations, particularly in Western vs other in Table 1, fairly high concentrations of phenol, hydro non-industrialized societies. For example, the microflora from quinone, catechol and benzenetriol are found in coffee. Lev individuals in developed countries have higher levels of Bac- els of hydroquinone in some herbal teas were estimated as teroides, whereas the flora from individuals from undeveloped high as 1%. Lower concentrations of phenol or hydroquinone countries exhibit higher counts of beneficial Lactobaci///'.13,14 have been measured in numerous vegetable-based products, Several proposed reasons for these differences include the alcoholic beverages, dairy products, green and black teas, sterility of processed western foods, and high meat intake in fruits, roasted nuts, honey, molasses, beef, and spices. western diets which results in a substitution of Lactobaci//i Table 1 Dietary sources of phenol, hydroquinone and catechol Source Phenola Hydroquinone Catechol Ref. Coffee Roasted beans Infusion Alcoholic beverages Red wine Beer Sherry Rum Teas Green or black Herbal (leaves of blueberry, cranberry, bearberry) Dairy products (milk, cheese, butter, whey) Roasted nuts (almonds, filberts, macadamia, peanuts) Fruits (apples, cherries, cranberries, blueberries, pears, tomatoes) Vegetable products (asparagus, broccoli, dried mushrooms, hydrolyzed soy protein, onion, rice cereal, roasted barley, roasted sesame seed, rye crisp bread, popcorn) Miscellaneous (honey, molasses, cocoa, beef, tamarind) 1.2-17 p.p.m. 30-40 p.p.m. 80-120 p.p.m. 0.2 p.p.m. -100 p.p.m. <0.02 p.p.m. detecteda <0.01 p.p.m. detected 0.5 p.p.m. detected detected detected <0.05 p.p.m. <0.1 p.p.m. <1% <0.1 p.p.m.b <0.1 p.p.m. detected detected 61,99-101 61,102 102,103 102,104 102 61,105 61,102 102,106 aCells left blank indicated no data. `Detected' means the study reported measuring the phenolic species, but did not quantitate the level. bUp to 110 p.p.m. of hydroquinone has been detected as arbutin in pears (see Table 2). Leukemia Editorial TA McDonald et al 15 Figure 3 Phenol production by gut bacteria. Data compiled from Bone et a!,54 Smith and MacFarlane,62 and Marteau et al.8 (which do not produce phenol) with high levels of Clostridia and Bacteroides populations (which produce phenol).13 Epidemiological studies have identified several risk factors for leukemia including consumption of high meat diets,10,11 use of antibiotics,12 and absence of breastfeeding.13-16 Each of these risk factors is associated with the potential to alter colonic microflora activity, composition, and resultant phenol production. It is interesting to speculate that phenol pro duction by the gut flora may be a contributing factor in these epidemiological associations. Individuals consuming high-beef diets have increased populations of phenol-producing anaerobic Bacteroides com pared to individuals on vegetarian diets.87,88 Comparisons of western and eastern populations indicated that individuals who consume a typical British and American high-meat diet have a higher ratio of anaerobic to aerobic microflora than persons from Japan, Uganda or India, whose diets are largely vegetarian.89 These findings were confirmed in a study of vol unteers who adopted a strictly vegan diet which led to a decrease in serum and urinary phenol and p-cresol concentrations.59 Upon returning to a conventional diet that included the addition of meat and eggs, the volunteers' urinary phenol and p-cresol concentrations correspondingly increased.59 Sub stitution of carbohydrate for protein may not be the only factor responsible for the reduction in phenol levels seen in veg etarians. Carbohydrate fermentation interferes with bacterial protein degradation90 and would, therefore, reduce the level of tyrosine metabolism in the gut. Antibiotic treatment is known to change intestinal microflora composition14 by suppressing the growth of some strains of bac teria while promoting the growth of others. For example, the administration of antibiotics is a factor responsible for the lower colonization rate of Lactobacillus in infants,91 while studies by Snydman and colleagues92 have shown the antimicrobial resist ance of Bacteroides fragilis. This resistance could lead to dis turbances in the intestinal flora, encouraging the overgrowth of these phenol-producing bacteria. Since the sequence in which bacteria populate the colon influences subsequent species' colonization and proliferation, it has been hypothesized that early colonization by Lactobacillus serves as a barrier against potentially pathogenic bacteria.14 In the absence of this barrier, other bacteria may flourish. Interestingly, breastfeeding also serves as another source of Lactobacillus,93 which do not produce phenol (Figure 3), and breastfeeding has recently been associated with a reduction in risk of childhood leukemia.15 More study into this possible link is needed. Hydroquinone from arbutin Arbutin, a glucose conjugate of hydroquinone (4-hydroxy-j8D-glucopyranoside), occurs naturally in many plant foods. Arbutin is readily hydrolyzed in the stomach to free hydroquinone, which is extensively absorbed through the gastro intestinal tract. Studies by Deisinger et al61 showed that wheat products and pears contain high levels of arbutin while the concentration of free hydroquinone is quite low (Table 2). They identified the highest levels of total hydroquinone in wheat germ (10.65 p.p.m.) and d'Anjou pears (15.1 p.p.m.). While lesser amounts of total hydroquinone are found in bev erages such as coffee, tea, and red wine (0.1 to 0.4 p.p.m.), these beverages may contribute significant amounts of hydroquinone to the diet, particularly in those who drink more than one cup (>200 ml) per day. Within 2 h of ingestion, volun teers fed a high arbutin-containing diet (784 to 1279 gg total hydroquinone) exhibited a five-fold elevation in plasma hydroquinone levels, increasing from a mean background level of 0.028 p.p.m. to 0.14 p.p.m.61 Likewise, significant increases in urinary hydroquinone levels were observed. Background levels of hydroquinone in the range of 0 to 100 gg/h surged to 700-1200 gg/h for up to 6 h following treatment.61 Leukemia Q) Editorial TA McDonald et al 16 Table2 Total arbutin and free hydroquinone (HQ) in commonly dative DNA damage, aneuploidy, chromosomal rearrange consumed foods and beverages (data from Deisinger et al61) ments, topoisomerase II inhibition, mitotic spindle disruption, Food Free HQ (p.p.m.) Total arbutin Serving size Average HQ and HQ (g) intake fog (p. p. m.) per serving) altered hematopoiesis and clonal selection. Thus, our hypoth esis, which synthesizes all these observations, is that back ground levels of phenol and hydroquinone are contributing factors to the development of some forms of leukemia in Wheat cereal 0.02 0.04 Whole wheat <0.21 bread Wheat germ <0.02 Pear Bosc <0.02 d'Anjou <0.05 Onion <0.01 Coffee 0.22 0.05 Tea <0.02 Red wine 0.5 1.0 0.1 2.0 0.4 10.7 3.6 3.8 0.7 15.1 11.7 0.5 0.3 0.04 0.1 0.02 0.4 25 12 3 180 180 85 200 200 180 23-28 19-29 21-43 558-810 612-4824 42.5 52-68 16-24 72 adults and children. An overview is presented in Figure 4. Predictions and critiques of the hypothesis If this model (Figure 4) is true, then several predictions can be made. For example, background rates of some forms of leuke mia should correlate with mean phenol and hydroquinone lev els in different populations. Such associations, however, may be overshadowed by genetic and environmental differences among the groups. The model predicts that high protein intake will be a risk factor for leukemia, an association that has been reported in some studies.10,11 High protein intake increases the Summary of hypothesis proportion of tyrosine and phenylalanine relative to low-protein diets, and also changes the composition of the gut flora towards The studies described above show that high background levels of phenol and hydroquinone are present in the blood of vir tually all individuals, but vary widely. The background levels more phenol-producing strains. Additional studies are needed to test this association and to determine if conversion of protein to phenol by the gut is an important factor. of phenol or hydroquinone stem from direct dietary ingestion, Our hypothesis predicts a wide inter-individual variability catabolism of tyrosine and other substrates by gut bacteria, ingestion of arbutin-containing foods, cigarette smoking, use in leukemia susceptibility among individuals because of widely differing gut flora composition, genetics, medicine and of some medicines, and environmental exposures to benzene tobacco use and, of course, large variations in diet. This may (Figure 4). Once inside the body, phenol is converted to explain why susceptibility to de novo leukemia has been so hydroquinone, primarily in the liver. Phenol, hydroquinone difficult to explain in persons without any obvious exposures and other phenolic species are transported via the blood to to leukemogenic agents, such as benzene and ionizing radi distant tissues including the bone marrow (Figure 4). Extensive ation. Specific predictions of our hypothesis include: (1) a high investigations into the mechanism(s) of leukemogenesis of intake of arbutin from the diet and the use of certain medi benzene indicate that hydroquinone, or hydroquinone in cines will be risk factors for developing leukemia; (2) inflam combination with phenol or other related phenolic metab matory bowel disease will be a risk factor in leukemia; and olites, are probably involved in cancer induction and pro gression. Indeed, hydroquinone and the other phenolic (3) null or low NQO1 activity or high myeloperoxidase activity will be risk factors. metabolites of benzene are associated with DNA adducts, oxi Probably the strongest argument against our hypothesis is that phenol and hydroquinone are not identified as carcino gens.94,95 Recently, the International Agency for Research on Cancer (IARC) evaluated the animal and human evidence for carcinogenicity, and in both cases concluded that phenol or hydroquinone was 'not classifiable as to its carcinogenicity to humans (group 3)'.94 In the case of phenol, the IARC con cluded that there was inadequate evidence in animals and in humans.94 Of the available studies of phenol-exposed work ers, all were accompanied by co-exposures to carcinogens. Phenol has been tested in one study of male and female rats and mice. An increased incidence of leukemia was reported in male rats treated with the lower dose, but not in the high- dose male rats, in mice or female rats. Phenol acted as a tumor promoter in tumor initiation-promotion studies using mouse skin. In the case of hydroquinone, the IARC concluded that there was inadequate evidence in humans and limited evi dence in animals for carcinogenicity. Several studies of photo graphic processing involving hydroquinone exposure have been reported. The IARC94 felt that only one of these studies provided enough information to demonstrate exposure. In that study a statistically significant increased incidence of malig nant melanoma was reported. Hydroquinone has been tested for carcinogenicity in two studies of rats and mice, inducing benign liver tumors in female mice of one study and male mice in another, benign kidney tumors in the male rats of both studies, and mononuclear cell leukemia in female rats of one study. The ability to detect cancer associations with hydro- Leukemia quinone may be difficult given the high background variation and intake from other sources. As described above, co-admin istration of phenol and hydroquinone reproduce many of the toxic effects observed following exposure to benzene. It may be that the redox cycling and synergistic effects associated with co-exposure are involved in leukemogenesis. No animal cancer study has co-administered phenol and hydroquinone. Studies have suggested that phenol and hydroquinone, either ingested or formed via the gut flora, will be rapidly con jugated and excreted, more so than phenol and hydroquinone formed from metabolism of benzene.96 This phenomenon is due to zonal distribution of conjugating enzymes within the liver.96 This fact may relate to the lack of clear associations between cancer induction and exposure to phenol or hydro quinone in humans or animals. It is true that phenol and hydroquinone are efficiently conjugated in the gut and liver by sulfotransferases, glutathione transferases and glucuronyl transferases. However, the observations of blood levels of free phenol and hydroquinone,59,61 and high levels of macromolecular adducts of benzoquinone (oxidized hydroquinone) in the blood of humans66 and blood and bone marrow of rod ents65,68 clearly indicate that a significant portion of ingested hydroquinone evades conjugation and is transported to distant tissues and is available for binding. Sulfatases, which cleave phenol and hydroquinone conjugates, may play a role in phenol and hydroquinone availability.17 A separate criticism is that we do not know for sure the carcinogenic mechanism of benzene and whether phenol and hydroquinone truly are the primary reactive metabolites. If there were a significant change in our understanding of the carcinogenic mechanism(s) of action for benzene, then our hypothesis would become invalid. As reviewed above, there is a large body of evidence to support the prevailing theory that phenol and hydroquinone are critical players in benzeneinduced leukemia. However, other possible mechanisms have been proposed. For example, benzene oxide, a reactive spec ies and the first oxidation product of benzene (Figure 1), has been recently shown to be somewhat stable in blood (half-life of 8 min).97 Moreover, benzene oxide adducts with proteins have been measured in rat and mouse bone marrow following administration of benzene.68 The relative importance of ben zene oxide to other metabolites is not known at this time. Also, other researchers have suggested that open-ring metab olites such as trans, trans-muconaldehyde may also play a role in benzene-induced hematotoxicity (reviewed in Ref. 98). The relative importance of these metabolites is believed to be small, but there is some uncertainty on this point. A significant drawback of the hypothesis is that it will be very difficult to test. We have noted in previous publi cations4,6,36 that there is wide inter-individual variability in susceptibility to hydroquinone-mediated hematotoxicity (eg from benzene exposure). This variability comes from genetic polymorphisms and differences in activity of key metabolizing enzymes (eg CYP2E1, NQO1 and myeloperoxidase), co exposures and other host factors. In this paper, we further describe the potential for significant variability in the back ground levels of phenol, hydroquinone and other phenolic species. This variability stems from differences in dietary and medicinal intake, gut flora composition and chemistry, smok ing and health status, and environmental exposures. Thus taken together, the numerous sources of variability in quinone-mediated hematotoxicity may reduce the power of a stu dy's ability to detect an association. Use of genetically homo geneous test animals may offer a solution; however, there Editorial TA McDonald et al currently does not exist a good animal model for benzeneinduced (ie quinone-mediated) leukemia. An attractive feature of our hypothesis is that it may explain why many people who have no known occupational exposures or significant smoking history develop leukemia. The hypothesis predicts that susceptibility to the disease would be related to diet, medicinal intake, genetics and gutflora composition. The latter two of these are largely beyond our control, and thus dietary modification and reduced use of medicines that elevate phenol levels may be the best inter vention strategies for lowering leukemia risk. Acknowledgements This work was supported by the California Environmental Pro tection Agency, the National Institute of Environmental Health Sciences (grants P42ES04705 and R01ES06721) and the National Foundation for Cancer Research. 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