Environmental Health Perspectives 105-9, 1997: Correspondence

Doubting Nongenotoxic Mechanisms of Renal Cancer: Comparing Apples and Oranges in the 2u-Globulin Hypothesis

Doubting accepted mechanisms in biology is not wrong per se, especially as the contrary would be the death blow to any form of advancement in research; however, doubting without good reason will consume precious energy and resources that could be spent on more fruitful and certainly more pressing matters in biological research.

Contrary to good judgment, it appears that some individuals believe that any biological mechanism, however meticulously established and proven with hard data and despite being commonly accepted, should be doubted when the mechanism in question tends to reduce the conception of risk associated with chemicals. This doubt, however, does not appear to stem from hard factual data contradicting the mechanism, but rather from misinterpretation of available data and, in some instances, from the misquotation of the original investigators' conclusions.

One recent example for doubting established and meticulously proven biological mechanisms is the ongoing discussion of the validity of the 2u-globulin-associated male rat-specific mechanism of renal cancer induction and its ramifications for human health risk assessment. A unique database (certainly in its size and detail when compared to other databases with similar importance for human health concerns) on practically all aspects of this mechanism has been established by numerous scientists from many different unaffiliated laboratories, repeated scientific discussion sessions, several thorough reviews (1-8), and a workshop specifically devised to query all aspects of the data that support the mechanism in question (9). Despite this, some scientists, in an unfathomable quest to differ, choose to ignore the obvious and continue to doubt the validity of this mechanism by throwing up new hypotheses (10) or interpretations of the present data (11). Such maneuvering is all the more reproachable as it is not constructive to risk assessment; it does not help to further the understanding of the currently accepted mechanisms; and foremost, the authors of such devil's-advocate papers have never presented new experimental data to support any of their new insights. In an effort to critically address some of the alternative hypotheses and interpretations presented by James Huff (11) in relation to the 2u-globulin mechanism, John Ashby, in a letter in Environmental Health Perspectives (12), asked Huff to relent from generalized statements and to "identify the chemicals to which he referred and present the relevant data for them in a focused paper."

Huff, in a reply in the same issue of EHP, rose to the occasion and tried to cement some of his statements and interpretations by specifically naming the chemicals (13). I feel that I must reply to the statements made by Huff in his letter. In order to facilitate the understanding of the following paragraphs, it is necessary for the reader to have insight into the current hypothesis of the etiology of 2u-globulin nephropathy and associated male rat renal tumors as well as the supporting mechanistic data.

Briefly, male rats as well as female rats, but not mice, hamsters, guinea pigs, rabbits, dogs, nonhuman primates, or humans synthesize low molecular weight proteins (approximately 18,000-20,000 daltons) called 2u-globulins (also known as A2u in earlier days). Synthesis of 2u-globulin is reported for female and male rats in the salivary, lachrymal, preputial, meiobian, and perianal glands (14,15). The hormonal regulation of 2u-globulin synthesis in each of these tissues is unique and, most importantly, not sex specific in the lachrymal, salivary, and preputial glands (15,16). These 2u-globulin isoforms are electrophoretically distinct from the major urinary protein, namely the hepatic form of 2u-globulin with a molecular weight of 18,700 daltons, which is exclusively synthesized by male rats. The 2u-globulin forms synthesized in small amounts by female rats are also excreted via the urine. However, these forms of 2u-globulin show distinct differences to the male rat forms of 2u-globulin, whether of nonhepatic or hepatic origin, suggesting that they are encoded by different genes (17).

The synthesis and high rate of excretion of the hepatic form of 2u-globulin, the affinity of some chemicals and/or their metabolites for binding to 2u-globulin, and the reduced enzymatic breakdown of the chemical- 2u-globulin complex in lysosomes of the proximal tubule epithelial cells (18) lead to an accumulation of these protein-chemical complexes in the renal cortex of male rats. Subchronic and chronic administration of chemicals was demonstrated to be mandatory for the development of nephrotoxicity and the formation of renal epithelial cell tumors in male rats (2-5). The renal tumors were demonstrated to evolve from a constantly elevated rate of proximal tubule epithelial cell proliferation resulting from the chemical- 2u-globulin complex-induced cell necrosis and subsequent cell regeneration (19-21). Due to the species- and sex-specific synthesis of this low molecular weight protein, nephropathy and kidney tumors are found in exposed male rats but not in female rats, 2u-globulin-deficient NBR male rats (21,22), either sex of mice, guinea pigs, rabbits, dogs, or nonhuman primates (1). Although humans and other species synthesize and excrete low molecular weight proteins via the urine, no interactions of these proteins with chemicals, shown to induce protein-mediated nephrotoxicity in the male rat, are observed (23,24). As a result of a joint effort of basic research conducted in both academic and industrial laboratories, the nongenotoxic mechanism underlying the genesis of these male rat-specific renal epithelial tumors is among the best characterized to date. Based on a 2-year review of this data set, the EPA ruled that the renal tumors detected in male rats following 2-year administration of nongenotoxic nephropathy-inducing compounds, such as unleaded gasoline or the orange juice component d-limonene, are of little relevance for human cancer risk assessment.

The EPA ruling on the 2u-globulin-mechanism and the associated simplifications and better understanding of at least one small part of the otherwise highly enigmatic processes involved in the etiology of cancer has apparently driven James Huff to question the mechanism described above by providing examples of so-called exceptions and discrepancies to the presently accepted mechanism as well as to make unfounded and scientifically unsound statements. For example, to show the weakness of the 2u-globulin mechanism hypothesis when it comes to extrapolating to the human situation, Huff states the following:

Importantly, two additional chemicals presumed to involve [ 2u]-kidney tumors (and thus are regarded by some as irrelevant to humans) have now been shown to be associated with kidney cancers in humans: gasoline [(25,26)] and trichloroethylene [(27,28)].

The studies on gasoline that Huff cited in the above context are epidemiological studies. This type of study looks at statistical associations between the increased occurrence of a disease and the exposure to gasoline (leaded gasoline). The weakness of such studies is that causal relationships and mechanisms can rarely be established as would be possible in highly specific animal studies, whereas their advantage lies in the fact that the endpoint of interest, tumors in humans, is inspected directly. However, the mere association of chemical exposure with increased occurrence of disease does not prove that there is a biological relationship. Most of the epidemiological studies performed on gasoline and the occurrence of human renal cancer have an additional important weakness in that they have not quantitatively assessed the actual exposure to volatile hydrocarbons. In addition, the studies were conducted with reference to leaded gasoline, thus adding the confounding factor of lead exposure, which is known to induce renal cancer in animal studies. Furthermore, in many studies, exposure assessment did not go beyond ascertaining that an individual had been employed by a petroleum company or in a refinery. Two of the studies (25,29) indicate a small increase in relative risk for kidney cancer in subgroups exposed to gasoline. However, in the studies by McLaughlin and co-workers (29,30) the most consistent association with renal cancer was found to be cigarette smoking, while in a subsequent more detailed analysis (31), no overall association was observed between renal cell cancer and employment in a range of occupations with potential exposure to petroleum products.

Siemiatycki et al. (25), a study specifically cited by Huff, looked at the risk for renal cancer in persons exposed to gasoline as well as jet fuel. No statistically significant risk for renal cancer was found for gasoline.

The only study reporting an elevated risk and an exposure-response relationship between kidney cancer and exposure to gasoline is the one published recently by Partanen et al. (26). These authors reviewed 672 cases of renal cell adenoma for a case-control study. Owing to poor participation, only 338 sets of cases and controls were ultimately included for analysis, thereby limiting the interpretability of the findings. Lifelong job histories were collected and translated into indicators of industry, occupation, and estimated occupational exposure. An elevated risk for kidney cancer was found to be associated with a history of white-collar occupations; the printing industry; the chemical industry; the manufacture of metal products; mail, telephone, and telegraph services; and iron and metal work. An elevated risk was associated with exposure to gasoline, and an exposure-response relationship was observed for increasing exposure to gasoline. However, a major confounding factor in this risk estimation was that the gasoline used in Finland, especially during the time relevant for exposure of the study group (1920-1968), contained copious amounts of tetraethyl-lead. The exposure to lead was associated with an increased risk for renal epithelial cell cancer, as was also confirmed by animal studies with lead acetate (32).

The association of renal cancer in humans with exposure to gasoline is most likely related to the tetraethyl-lead content of the gasoline and not to the gasoline (volatile hydrocarbons) itself, as was also clearly pointed out by Partanen et al. (26). Therefore, the statement by Huff, that "gasoline had been shown to be associated with kidney cancer in humans" is a misrepresentation of the scientific facts.

With regard to trichloroethylene, Huff admits that it does not induce the 2u-globulin-associated nephropathy; therefore, this example is irrelevant and a misrepresentation of facts. Huff tries to back his statement by adding that a chemically similar compound (perchloroethylene), which appears to be linked (no proven causality) with human renal cancer, was demonstrated to induce 2u-globulin nephropathy and renal tumors in male rats. To substantiate this statement, he quotes Melnick (10), a paper that does not provide new data but selectively quotes Green et al. on perchloroethylene (33). Green et al. (33) clearly state that the tumors observed in the male rat kidney appear to be related to a combination of at least two mechanisms: genotoxicity from the �-lyase pathway and the 2u-globulin mechanism. However, Green et al. (33) also found that even the genotoxic mechanism is also species and sex specific and is of little relevance to humans. Such synergy between a genotoxic agent (initiator) and a promoting influence (in this case the 2u-globulin mechanism via enhanced cell proliferation) is well established not only in renal carcinogenesis. That is exactly why the EPA, in its ruling, restricted the relevance of the 2u-globulin mechanism in conjunction with renal tumors occurring only in male rats to nongenotoxic chemicals. This emphasizes that we cannot define a compound as either being an 2u-globulin nephropathy inducer or not; we have to understand the etiology of the tumors induced by a given chemical to detect whether only one or several mechanisms are involved in its genesis.

Thus, neither the epidemiological studies on leaded gasoline nor any of the polychlorinated aliphatics cited by Huff lend themselves to questioning the 2u-globulin mechanism. The available human epidemiological studies and the animal experimental studies were misinterpreted and misrepresented by Huff. This distorts the factual content of the data presented and the ramifications of these facts for human health.

Some of the other chemicals Huff mentioned as being examples that contest the 2u-globulin mechanism were gabapentin, lindane, decalin, trimethylpentane (unleaded gasoline), dimethyl methylphosphonate, t-butyl alcohol, and hexachlorobenzene. I will not discuss all of the chemicals in detail, although none of them are examples that would contest the 2u-globulin mediated mechanism. Instead, I would like to point out some important facts that Huff overlooked. The five examples selected for discussion are representative.

As pointed out by Huff, gabapentin induces an accumulation of 2u-globulin in the proximal tubule epithelial cells of male rats and, after prolonged administration (up to 2, 7, and 14 days) and application of 2,000 mg/kg body weight (bw), even produces some cell necrosis and cell shedding (34). No increase in renal cancer was observed after 2 years of gabapentin administration (34). However, what Huff did not find necessary to point out is that, in all cases where chemicals induced 2u-globulin nephropathy and renal tumors, increased amounts of proteinaceous intratubular casts, especially at the cortico-medullary junction, and, consequently, increased cell regeneration were observed (20,21,35,36). Indeed, chronic increased cell regeneration is the hallmark of chemically induced renal tumors via the 2u-globulin mechanism (1). The question then arises whether the prolonged gabapentin administration induced increased numbers of proteinaceous intratubular casts, especially at the cortico-medullary junction, and consequently enhanced rates of cell regeneration. A closer look at the paper of Dominick et al. on gabapentin (34) clearly indicates that no dose response was observed in the number of casts or in the severity of renal tubular regeneration at 13 weeks of exposure (doses up to 3,000 mg/kg bw). This data clearly indicates that the severity of 2u-globulin accumulation and subsequent cell necrosis and proliferative regeneration was not high enough to allow for formation of renal tumors within the 2 years of gabapentin exposure. Dominick et al. (34) also found foci of atypical hyperplasia in kidneys of male rats treated with 1,000 and 2,000 mg/kg bw at termination of the 2-year bioassay. Atypical tubules and atypical hyperplasia have been recognized as precursors of renal adenoma and carcinoma (19-21,32). The reported number of atypical foci was extremely low in the Dominick et al. study (34). In summary, the present database on gabapentin does not disprove the 2u-globulin-associated mechanism of renal carcinogenesis, but rather demonstrates that chemicals that cause 2u-globulin-mediated renal cancer must bind to 2u-globulin with high affinity to produce chronic elevated cell necrosis and subsequent cell regeneration. Indeed, this view is also shared by Dominick et al. (34) who specifically stated in their paper that

this study suggests that proximal tubular epithelial injury and regeneration that occur in this [a2u-globulin] nephropathy may need to reach a critical threshold to effectively promote renal neoplasia.

The second compound that Huff mentioned as being one of the compounds that does not induce renal tumors in male rats despite being a typical 2u-globulin compound is lindane. It is indeed true that lindane and its metabolites can bind to 2u-globulin and induce the associated nephropathy in male rats during acute exposures (37). Lindane (and/or its metabolites) has been shown in the 2-year carcinogenicity bioassay to increase the incidence of benign and malignant neoplasms in endocrine organs (pituitary, adrenal, and thyroid) of both sexes; however, a high incidence of ovarian carcinoma was also noted (38). Moreover, male rats treated with lindane at 32 and 64 mg/kg bw for 2 years presented with severe testicular atrophy. It is thus quite reasonable to assume that the testosterone level in these rats, as a consequence of testicular atrophy, was low. However, testosterone is extremely important for maintaining a high level of liver-derived 2u-globulin, as will be outlined below. Indeed the male rat major urinary protein (the hepatic form of 2u-globulin) is encoded by a family of 25-30 copies of highly homologous (less than 5% divergence in the nucleic acid sequence) genes within the haploid genome, each made up of 7 exons and clustered on chromosome 5 (39,40). The expression of this 2u-globulin gene family is regulated by a complex interaction of testosterone, glucocorticoids, insulin, growth hormone, and thyroid hormone (4). The synthesis of hepatic 2u-globulin is under androgenic control, as evidenced by changes in androgen sensitivity and androgenic induction of 2u-globulin synthesis, which begins at puberty (approximately 40 days in rats), peaks at about 80 days, and then steadily declines in expression level only to cease at senescence (i.e., 750-800 days of age) (41). The maturational rise in 2u-globulin synthesis and its decline during senescence are associated with corresponding changes in the expression of the androgen receptor gene in the liver and thus with changes in the steady-state level of the 2u-globulin mRNA (42). Transcriptional activation of the 2u-globulin gene coincides with its attachment to the nuclear matrix, while conversely, senescent-associated transcriptional inactivation is accompanied by matrix detachment of this gene (40).

Estrogenic steroids are very effective repressors of 2u-globulin synthesis (43). Indeed, estrogen receptor complexes were shown to bind to the promoter region of the 2u-globulin genes (44) and were thus able to repress the expression of these genes. It is thus not surprising that female rats, with their inherently high levels of estrogen, do not synthesize hepatic 2u-globulin or its corresponding mRNA, despite having the entire complement of hepatic 2u-globulin genes (16,45,46). Ovariectomized female rats have a very low background level of 2u-globulin mRNA (47), and ovariectomy in conjunction with androgen treatment of female rats results in an increase of 2u-globulin mRNA as well as protein product (45,48).

Male rats after puberty, on the other hand, were shown to have high levels of estrogen sulfotransferase, an enzyme able to inactivate estrogen activity and thus maintain androgen sensitivity of the male rat liver during the peak phase of 2u-globulin synthesis (49).

Based on the assumption that high levels of 2u-globulin could not have been maintained in male rats treated with high doses of lindane in the 2-year bioassay, due to testicular atrophy and thus a lack of testosterone, it becomes obvious why renal tumors did not develop in lindane-treated male rats. Lindane, therefore, is not a compound that would question the 2u-globulin-mediated mechanism of male rat renal tumors as Huff believes, but rather it supports the mechanism.

The question that remains is why lindane was reported to induce 2u-globulin nephropathy. Indeed, 2u-globulin nephropathy was observed in male rats treated with lindane 10 mg/kg bw day for 5 days (37). The levels of 2u-globulin and the resistance to lysosomal breakdown of the 2u-globulin-lindane complex was high enough that this nephropathy could be observed under acute exposure. In contrast, no hyaline droplets, nephropathy, or renal casts were observed in the rats dosed with 32 and 64 mg/kg bw for 2 years in the cancer bioassay (38).

With regard to risk assessment, lindane could have not been regulated under EPA's ruling on 2u-globulin nephropathy-inducing compounds, based on the fact that no renal tumors were detected in male rats and that tumors in other organs in both male and female rats were induced in the 2-year bioassay. Lindane, however, may well be an example of the problems associated with the maximum-tolerated-dose (MTD) approach in the 2-year cancer bioassays. It is indeed conceivable that male rats would have developed renal tumors in the 2-year bioassay if the chronic dose had been at a concentration that would not have induced testicular atrophy and thus not have impeded testosterone production. In conclusion, lindane does not provide a good example to substantiate theories, but rather it exemplifies how a lack of understanding of mechanisms can lead to misconceptions.

The latter statement also holds true for decalin. Indeed, Huff states that

decalin is probably the first chemical for which no empirical connection could be made between induced hyaline droplet-[ 2u-globulin] nephropathy syndrome and cancer of the renal epithelial cells.

Huff cites Gaworski et al. (50), who quite clearly demonstrate all classical symptoms of an 2u-globulin-mediated mechanism; this 90-day study could not have promoted the necessary number of preneoplastic lesions that would have progressed to veritable tumors within the 19-month post-exposure observation period. Indeed, the 2u-globulin-mediated mechanism of renal tumor induction is a pure promotional process. Thus, a chronic stimulus (chronic cell necrosis and subsequent chronic cell regeneration) is necessary for a low induced incidence of renal tumors to be observed at the end of a 2-year bioassay. Furthermore, the promotional effect of the 2u-globulin mechanism is weak, leading to low renal tumor incidences in 2-year rat bioassays (up to 25%) (51), while other presumably nongenotoxic compounds, e.g., ochratoxin A, lead to high renal tumor incidences (up to 87%) in both sexes of rats (52). Therefore, Huff's statement that

at the end of the post-exposure period, male rats exhibited dose-related chronic progressive necrosis with accentuated tubular degeneration, medullary mineralization, and papillary hyperplasia; however, no tumor of the kidney was reported for male rats or for any other sex-species-exposure group

is incorrect. Indeed, mentioning chronic progressive necrosis and tubular degeneration in conjunction with decalin treatment is misleading in this context. These symptoms are characteristic for aging rats (53), but can be exaggerated in a dose-related fashion following exposure to chemicals (19). However, there is no distinct association or causality between the occurrence lesions of chronic progressive necrosis (also known as chronic progressive nephropathy) and the etiology of 2u-globulin-chemical complex-mediated renal tumors. Therefore, insinuating that in the case of decalin, despite the presence of chronic progressive necrosis, no tumors related to 2u-globulin were observed is a misconception that unnecessarily leads to misunderstandings. Unfortunately no 2-year cancer bioassay has been conducted to date on decalin. It is hoped that, as Huff pointed out, the NTP will conduct a 2-year bioassay with decalin using F344 and the 2u-globulin-deficient NBR rats in the near future. I am quite confident that this study, as many others before, will substantiate the currently accepted mechanism.

The two last compounds that are worth discussing are trimethylpentane and hexachlorobenzene. Trimethylpentane (TMP) was demonstrated to bind to 2u-globulin (54-57) and to induce 2u-globulin nephropathy in male rats (36,58). Prolonged treatment of male rats with TMP exacerbates the nephropathy and increases regenerative cell proliferation in a dose- dependent manner (36,59,60). NBR rats lacking 2u-globulin do not produce protein accumulation or nephropathy following treatment with TMP (22). TMP was also demonstrated to be a renal tumor promoter (via the 2u-globulin-mediated mechanism) in a classic initiation-promotion study by Short et al. (19). This fact stands out against Huff's statement that TMP had been demonstrated not to induce renal tumors in a 2-year study. The literature cited, which back the 2u-globulin-mediated mechanism, appeared in peer-reviewed journals and thus were open for scientific scrutiny. On the contrary, Huff's statement is backed by a personal communication on a bioassay of which the experimental conditions and the actual resulting data are not available; these studies by the Instituto Ramazzini have not been duplicated by another laboratory using a different strain of rats, whereas the studies supporting the 2u-globulin-mediated mechanism have been reproduced in various laboratories. In this, Huff does not seem to be carefully weighing the evidence.

Hexachlorobenzene (HCB) was shown to induce 2u-globulin nephropathy in male rats but not in female rats, even when treated for 50 days at the highest dose (61). The statement by Huff that "hexachlorobenzene (HCB) is an example of a chemical that induces the [ 2u-globulin] nephropathy syndrome in male and female Sprague-Dawley rats..." (61) is a misrepresentation of facts. Female rats do not present with the 2u-globulin nephropathy syndrome in any case so far discussed. Furthermore, female rats did not present with any form of renal pathology, of whatever etiology, in the 50-day study by Bouthillier (61). It is true, however, that both sexes of rats treated with HCB presented with hepatomas (male, 11/56; female, 35/56), hepatocellular carcinomas (male, 4/56; female, 48/56), bile duct adenomas (male, 2/56; female, 29/56), and renal adenomas (male, 42/56; female, 15/56) (62). The incidences of renal adenomas were decisively higher in male than in female rats. The overt increase in liver carcinomas and bile duct tumors in both sexes of Sprague Dawley rats indicates that several mechanisms are involved in the etiology of liver, bile duct, and renal tumors, one of them most likely being a genotoxic mechanism. Hexachlorobenzene, therefore, is not an example that would contest the 2u-globulin hypothesis. Huff states that "HCB is the first chemical identified that exhibits the [ 2u-globulin] nephropathy sequelae, and induces renal cell tumors in both genders of rats." This statement is a misconception and misrepresentation of facts. In this context, it is also wrong and misleading of Huff to insinuate that HCB would have been regulated under the EPA ruling discussed here. Indeed, the EPA ruling on 2u-globulin-inducing compounds clearly concerns compounds only that 1) are nongenotoxic; 2) produce renal tumors in male rats and in no other sex, species, or organ; and 3) induce the 2u-globulin nephropathy syndrome. Hexachlorobenzene does not fulfill two of the above categories and should therefore not be given as an example in this context.

In conclusion, I would like to emphasize that one needs at least as excellent a database to disprove a currently accepted mechanism as was used to establish the mechanism in the first place. Questioning for queries sake is not helpful, but is rather hindersome to those scientists that excel in distinguishing those mechanisms that are relevant to humans and can be predicted via animal experimentation from those that are specific to the surrogate animals only. In his letter in Environmental Health Perspectives, James Huff was clearly not able to disprove the currently accepted mechanism; I challenge him to produce some experimental data to substantiate his hypothesis.

Daniel R. Dietrich
University of Konstanz
Environmental Toxicology Laboratory
Konstanz, Germany

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42. Song CS, Rao TR, Demyan WF, Mancini MA, Chatterjee B, Roy AK. Androgen receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging, and calorie restriction. Endocrinology 128:349-356 (1991).

43. Roy AK, McMinn DM, Biswas NM. Estrogenic inhibition of the hepatic synthesis of alpha 2u globulin in the rat. Endocrinology 97:1505-1508 (1975).

44. Van Dijck P, Verhoeven G. Interaction of estrogen receptor complexes with the promoter region of genes that are negatively regulated by estrogens: the alpha 2u-globulins. Biochem Biophys Res Commun 182:174-181 (1992).

45. Sippel AE, Feigelson P, Roy A. Hormonal regulation of the hepatic messenger RNA levels for alpha2u globulin. Biochemistry 14:825-829 (1975).

46. Sippel AE, Kurtz DT, Morris HP, Feigelson P. Comparison of in vivo translational rates and messenger RNA levels of alpha2u globulin in rat liver and Morris hepatoma 5123 D. Cancer Res 36:3588-3593 (1976).

47. Chatterjee B, Hopkins J, Dutchak D, Roy AK. Superinduction of alpha2u-globulin by actinomycin D: evidence for drug-mediated increase in A2u mRNA. Proc Natl Acad Sci USA 76: 1833-1837 (1979).

48. Roy AK, Neuhaus OW. Androgenic control of a sex-dependent protein in the rat. Nature 214:618-620 (1967).

49. Mancini MA, Song CS, Rao TR, Chatterjee B, Roy AR. Spatio-temporal expression of estrogen sulfotransferase within hepatic lobule of male rats: implication of in situ estrogen inactivation in androgen action. Endocrinology 131:1541-1546 (1992).

50. Gaworski CL, Haun CC, MacEwen JD, Vernot EH, Bruner RH, Amster RL, Cowan MJ Jr. A 90-day vapor inhalation toxicity study of decalin. Fundam Appl Toxicol 5:785-793 (1985).

51. NTP. Toxicology and Carcinogenesis Studies of d-Limonene (CAS No. 5989-27-5) in F344/N Rats and B6C3F1 Mice (Gavage Studies). TR-347. Research Triangle Park, NC:National Toxicology Program, 1990.

52. NTP. Toxicology and Carcinogenesis Studies of Ochratoxin A (CAS No. 303-47-9) in F344/N Rats (Gavage Studies). TR-358. Research Triangle Park, NC:National Toxicology Program, 1989.

53. Montgomery CA, Seely JC. Kidney. In: Pathology of the Fischer Rat (Boorman GA, Eustis SL, Elwell MR, Montgomery CA, MacKenzie WF, eds). San Diego:Academic Press, 1990;127-153.

54. Borghoff SJ, Lagarde WH. Assessment of binding of 2,4,4-trimethyl-2-pentanol to low-molecular-weight proteins isolated from kidneys of male rats and humans. Toxicol Appl Pharmacol 119:228-235 (1993).

55. Borghoff SJ, Strasser JJ, Charbonneau M, Swenberg JA. Analysis of 2,4,4-trimethyl-2-pentanol (TMP-OH) binding to male rat kidney 2u-globulin (A2u) and other proteins. Toxicologist 8:135 (1988).

56. Borghoff SJ, Upton PB, Swenberg JA. Characteristics of 2,4,4-trimethyl-2-pentanol (TM-POH) binding to A2u-globulin and other compounds that cause protein droplet nephropathy. Toxicologist 9:79 (1989).

57. Lock EA, Charbonneau M, Strasser J, Swenberg JA, Bus JS. 2,2,4-Trimethylpentane-induced nephrotoxicity. II. The reversible binding of a TMP metabolite to a renal protein fraction containing alpha 2u-globulin. Toxicol Appl Pharmacol 91:182-192 (1987).

58. Charbonneau M, Lock EA, Strasser J, Cox MG, Turner MJ, Bus JS. 2,2,4-Trimethylpentane-induced nephrotoxicity. I. Metabolic disposition of TMP in male and female Fischer 344 rats. Toxicol Appl Pharmacol 91:171-181 (1987).

59. Short BG, Burnett VL, Swenberg JA. Elevated proliferation of proximal tubule cells and localization of accumulated alpha 2u-globulin in F344 rats during chronic exposure to unleaded gasoline or 2,2,4-trimethylpentane. Toxicol Appl Pharmacol 101:414-431 (1989).

60. Borghoff SJ, Youtsey NL, Swenberg JA. A comparison of European high test gasoline and PS-6 unleaded gasoline in their abilities to induce alpha2u-globulin nephropathy and renal cell proliferation. Toxicol Lett 63:21-33 (1992).

61. Bouthillier L, Greselin E, Brodeur J, Viau C, Charbonneau M. Male rat specific nephrotoxicity resulting from subchronic administration of hexachlorobenzene. Toxicol Appl Pharmacol 110:315-326 (1991).

62. Ert�rk E, Lambrecht RW, Peters HA, Cripps DJ, Gocmen A, Morris CR, Bryan GT. Oncogenicity of hexachlorobenzene. In: Hexachlorobenzene: Proceedings of an International Symposium (Morris CR, Cabral JRP, eds). IARC Scientific Publications No 77. Lyon:International Agency for Research on Cancer, 1986;417-423.

The Relevance of Mechanistic Data to the Interpretation and Extrapolation to Humans of Rodent Carcinogenicity Data

I refer to Huff's response (1) to my letter to EHP regarding the adequacy of the 2u-globulin nephropathy mechanism of tumor induction in male rats (2) and to the reponse by Dietrich (3) to the points made by Huff (1). Pursuit of the mechanisms by which chemicals are toxic to mammals can be justified in two ways. The first is that the acquisition of such knowledge may increase our ability to prevent or attenuate human diseases. The second is that mechanistic data can contribute significantly to our ability to differentiate significant and trivial chemical hazards and to assess likely risks to humans. This latter justification can be illustrated by the following statement: if all chemicals that are mutagenic in vitro were mutagenic and carcinogenic to humans, and if all rodent carcinogens posed an equal carcinogenic hazard to humans, then there would be no role for mechanistic studies. The fact that these statements are self-evidently untrue therefore provides the main justification for the conduct of mechanistic studies.

The value of the results of mechanistic studies aimed at differentiating hazards should be judged solely in terms of the extent to which they either increase or decrease the level of concern over the potential hazard posed to humans by exposure to the substance under study. Within this context, simple experiments, such as confirming that an in vitro mutagen is also mutagenic in vivo or establishing the effect that changing the route of exposure, or the test species, has on the carcinogenicity of a substance, qualify as mechanistic studies. However, the term is usually reserved for investigations that provide a framework within which the rodent toxicity of a substance can be explained and extrapolated to humans.

Loose use of the term mechanistic studies can lead to confusion when the derived results offer no guidance on human hazard assessment. For example, it may be interesting to establish that nitrogen mustard is clastogenic by virtue of its ability to bind covalently with DNA, while the similar activity of etoposide is due to its ability to inhibit topoisomerase II enzymes, but such knowledge is of no value for human hazard assessment in the absence of data indicating that one of these mechanisms of clastogenesis is more relevant to human hazard assessment than the other. This single example indicates that when mechanistic data are invoked to qualify the hazard of a rodent carcinogen, there is a need to discuss the context of those data and to make clear how they should alter our perception of the human hazard implied by the initial test data.

To date, there are no examples of a mechanism of cancer induction in rodents that is rigorously established and uniformly accepted as being of no relevance to humans. Further, given the potential for occasional high accidental exposures to chemicals and the existence of human polymorphisms among the enzymes that activate or detoxify chemicals, and among the enzymes and processes that maintain genetic integrity, such an example is unlikely ever to be documented. Nonetheless, there are several proposed mechanisms of cancer induction (rodent tumor etiologies) and tumor types (e.g., B6C3F ₁ mouse liver tumors) that carry a high level of certainty regarding their probable irrelevance to humans. The central issue therefore becomes whether a given level of certainty is acceptable or not. Whichever conclusion is reached in such cases, a compromise will have been made in the process. So, for example, if a chemical induces specifically renal or thyroid gland tumors in a lifetime rat carcinogenicity bioassay and if those tumors are preceded by the induction of 2u-globulin-mediated nephropathy or thyroid-stimulating hormone-mediated thyroid follicular cell proliferation, respectively, then a decision will have to be made regarding the relevance of those tumor etiologies to humans.

The differences evident in the viewpoints of Huff (1) and Dietrich (3) are due to both different starting premises regarding the value of attempting to separate mechanisms of chemical carcinogenesis and differences in their use and interpretation of the available mechanistic data for the 2u-globulin-mediated nephropathy mechanism. Those differences are of interest at two levels. First, they lead to identification of the key data by which to assess whether or not humans will be subject to this particular rodent tumor etiology. Uncertainties over the sufficiency and the significance of those data remain a valid subject for future research. Second, the different viewpoints expressed confirm that we have some way to go before we can all share a common motivation for undertaking mechanistic studies. Put simply, we will make no useful progress until we can share the motivation of attempting to improve human hazard assessments within the limited confines of the resources available for the task, mindful that compromises will have to be made if we are to distinguish, for example, the human hazards posed by exposure to the rodent kidney carcinogens limonene and diethyl nitrosamine. That motivation will lead only to rational and balanced discussion of the relevant peer-reviewed data. As summarized in the old Chinese adage, "if you want to find the truth about an issue, don't take either side." Attempts to guard the scientific high ground and to search for certainty, ostensibly on behalf of less well-informed colleagues, or attempts to dismiss rodent carcinogenicity data by reference to insecure and preliminary mechanistic constructs must be equally resisted.

The success or failure of the margin of exposure (MOE) approach to nongenotoxic carcinogen hazard assessment proposed by the U.S. EPA will depend largely on the extent to which such common ground can be found and secured in the future.

John Ashby
Zeneca Central Toxicology Laboratory
Alderley Park, Cheshire, United Kingdom

References

1. Huff J. Response: 2�-globulin nephropathy, posed mechanisms, and white ravens [letter]. Environ Health Perspect 104:1264-1267 (1996).

2. Ashby J. 2u-Globulin nephropathy in white ravens [letter]. Environ Health Perspect 104: 1264 (1996).

3. Dietrich D. Doubting nongenotoxic mechanisms of renal cancer: comparing apples and oranges in the 2u-globulin hypothesis [letter]. Environ Health Perspect 105:898-902 (1997).

2u-Globulin Nephropathy and Ravens: Do Ravens of a Different Feather Flock Together?

The scientific community is well served by the continued interest in mechanisms of 2u-globulin nephropathy and its potential relationship to nephrocarcinogenesis in rodents. The article by Huff (1), commented on by Ashby (2), and with a subsequent reply by Huff (3) sparked a renewed polemic. In addition, a letter by Dietrich (4) further fuels this scholarly debate.

Having contemplated the diverse opinions from these authors, we are concerned that some of the statements based on published data from our laboratories (5-7) might be viewed in different context than originally presented, leading to potential misinterpretations. To clarify these statements from other scientists, we feel that it is necessary to touch upon the following facts.

Over the last decade, we have developed extensive preclinical safety evaluation data to support the human use of gabapentin. Gabapentin is a safe and effective drug for epilepsy based on rigorous review by regulatory agencies worldwide. From these studies, risk benefit comparisons were made between animal and human dosages. Wistar rats in the 2-year carcinogenicity bioassay showed significantly higher gabapentin exposures (as measured by plasma concentrations and area under the curve) than humans at therapeutic exposures.

When exposed to gabapentin for 2 years at 10-15 muItiples of the human exposure, rats developed mild 2u protein accumulation as judged by histomorphologic examination. After 13 weeks of exposure, the incidence of 2u-globulin accumulation was similar across the treatment and control groups, and the degree of severity was higher in the treated groups. The extent of 2u protein accumulation in the kidneys of male rats treated with gabapentin diminished after 52 weeks, whereas the severity of spontaneous chronic nephropathy in treated male rats relative to controls increased from 52 and 104 weeks.

As a result of the current discussion in the literature (3), the kidney specimens were reanalyzed for the hallmarks of 2u-globulin nephropathy sequelae, namely the presence of granular casts and linear mineralization (8). These changes were not observed in male rats receiving 250, 1,000, or 2,000 mg/kg of gabapentin for 2 years. In terms of proliferative effects, there were 3 foci of atypical hyperplasia of renal tubules: 2/50 were found in male rats at 1,000 mg/kg and 1/50 male rats at 2,000 mg/kg of gabapentin. These kidney examinations in the 2-year study included four sections (two from each kidney) from every animal in the group (50/sex/group), amounting to 1,600 kidney sections in the 2-year rat study. This standard procedure seems to adequately sample the kidney.

Reference was made to renal accumulation of gabapentin and to its ability to bind 2u-globulin. Metabolic disposition data revealed no differences in the accumulation of gabapentin in the kidney of male and female rats. Gabapentin distributes rapidly to the kidney in both sexes, and approximately 10-12% of the dose is found at 2-hr post-dose. The elimination decay is rapid, and 0.20-0.14 gabapentin radioequivalents in micrograms per gram are found at 12 hr post-dose. Full profile toxicokinetic studies have shown linear kinetics without accumulation, and elimination follows rapid clearance from the plasma and organ compartments. Recoveries in the mass balance studies yielded 99% or more of the administered dose.

Why such a diverse series of chemicals causes a putatively similar microglobulin nephropathy with (i.e., unleaded gasoline) and without (i.e., gabapentin) nephrocarcinogenesis is intriguing.

Evaluation of the gabapentin data does not prove or disprove that 2u-globulin accumulation precedes, promotes, or causes renal cancer in rats over a 2-year constant exposure at toxicity-limiting doses. We propose that although morphofunctional features may appear similar for all documented cases of this xenobiotic-induced nephropathy, different mechanisms may be operative. Alternatively, a structural comparison of the 2u microglobulin species would assert this microglobulin not to be the same in all cases when induced by the diverse chemicals. The work by Hildebrand et al. (9) would indicate some support to this notion. In the case of gabapentin, in which we have substantial data on hand, it is difficult to advocate existing mechanisms because of the drug's lack of metabolism or biotransformation, lack of serum protein binding, or lack of significant organ accumulation. It remains unanswered whether data on cell proliferation or specific 2u-globulin binding to renal tubule proteins would substantiate current mechanisms. Clearly new data, rather than new assumptions, will give fresh insight to our understanding of the 2u nephropathy puzzle.

Felix A. de la Iglesia
Alexander W. Gough
Robert E. Sigler
Pathology and Experimental Toxicology
Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company
Ann Arbor, Michigan

References

1. Huff J. Mechanisms, chemical carcinogenesis, and risk assessment: cell proliferation and cancer. Am J Ind Med 27:293-300 (1995).

2. Ashby J. 2�-Globulin nephropathy in white ravens [letter]. Environ Health Perspect 104:1264 (1996).

3. Huff J. Response: 2�-Globulin nephropathy, posed mechanisms, and white ravens [letter]. Environ Health Perspect 104:1264-1267 (1996).

4. Dietrich D. Doubting nongenotoxic mechanisms of renal cancer: comparing apples and oranges in the 2u-globulin hypothesis [letter]. Environ Health Perspect 105:898-902 (1997).

5. Sigler RE, Gough AW, de la Iglesia FA. Pancreatic acinar cell neoplasia in male Wistar rats following 2 years of gabapentin exposure. Toxicology 98:73-82 (1995).

6. Fowler ML, Sigler RE, de la Iglesia FA, Reddy JK, Lalwani ND. Absence of Ki-ras mutation in exocrine pancreatic tumors from male rats chronically exposed to gabapentin. Mutat Res 327:151-160 (1995).

7. Dominick MA, Robertson DG, Bleavins MR, Sigler RE, Bobrowski WF, Gough AW. 2�-Globulin nephropathy without nephrocarcinogenesis in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid. Toxicol Appl Pharmacol 111:375-387 (1991).

8. Swenberg JA, Short B, Borghoff S, Strasser J, Charbonneau M. The comparative pathobiology of 2�-globulin nephropathy. Toxicol Appl Pharmacol 97:35-46 (1989).

9. Hildebrand H, Hartmann E, Popp A, Bomhard E. Quantitation of 2-microglobulin after administration of structurally divergent chemical compounds. Arch Toxicol 71:351-359 (1997).

Weight of Evidence Versus Weight of Speculation to Evaluate the 2u-Globulin Hypothesis

In response to a challenge from John Ashby (1) to identify chemicals that demonstrate inconsistency with the 2u-globulin nephropathy/rat kidney cancer hypothesis, James Huff (2) described the responses of seven relevant chemicals in an accompanying correspondence. Evidently that response hit a sensitive nerve in Daniel Dietrich (3), resulting in his needless attack on those who profess different views on this issue.

Dietrich claims that the 2u-globulin mechanism has been "established and meticulously proven" and chastises those who "choose to ignore the obvious" by questioning the validity of this "accepted mechanism." To Dietrich, those who doubt this hypothesis are guilty of "misinterpretation of available data." Would Dietrich similarly criticize Short et al. (4), including his own mentor James Swenberg, for acknowledging that "the exact mechanism of cellular damage in 2u [globulin] nephropathy is unknown" or the EPA Risk Assessment Forum (5) for concluding that the 2u-globulin syndrome "should be considered a satisfactory working hypothesis but not a proven mechanism of action to describe renal tubule cancer in male rats exposed to CIGA" [chemicals that induce 2u-globulin accumulation]?

Rather amusing in this debate is that Dietrich does exactly what he wrongfully accuses Huff of doing, namely, misinterpreting data and misrepresenting various study findings to support his own viewpoint. We chose to use only three of the many distortions in Dietrich's letter for illustration. First, Dietrich claims that Partanen et al. (6) attributed the elevated risk of renal cell cancer in humans exposed to gasoline "to the tetraethyl-lead content of the gasoline and not to the gasoline (volatile hydrocarbons) itself." That is wrong. Partanen et al. (6) actually wrote that "our results suggest that some hydrocarbon constituent(s) or additive(s) of gasoline are conducive to renal cell cancer in humans," and although "tetraethyl lead is capable of dermal entrance . . . the amount of lead intake is probably low in the jobs with typical dermal exposure to gasoline." Furthermore, a recent epidemiological study of workers exposed to gasoline vapors (service station workers in the Nordic countries) found a 30% excess risk of kidney cancer with no excess risk of leukemia (7). The kidney cancer findings in humans exposed to gasoline vapors correspond to previously reported kidney cancer findings in male rats, which Dietrich ironically dismisses as irrelevant to human risk because of 2u-globulin accumulation.

Second, Dietrich attempts to explain the nonconcordance between lindane-induced 2u-globulin nephropathy and the lack of a male rat kidney tumor response by speculating about relationships among exposure to lindane, testicular atrophy, serum testosterone levels, and 2u-nephropathy. Dietrich and Swenberg (8) reported that a daily dose of 10 mg/kg lindane induces a "marked increase in 2u-globulin" in the kidney of male F344 rats. The National Cancer Institute's carcinogenicity feed study on lindane found no increase in kidney cancer or testicular atrophy in dosed rats compared to controls (9). Conversely, in a separate review of that study, Reuber (10) reported that the incidences of moderate and severe testicular atrophy were increased in the 472 ppm dose group (equivalent to 20 mg/kg/day) but not in the 236 ppm dose group (equivalent to 10 mg/kg/day) and that the incidence and severity of chronic renal disease, but not kidney cancer, was increased in both dose groups of male rats. Thus, lindane did not induce kidney tumors in male rats at a dose that caused a marked increase in renal 2u-globulin accumulation and that was not associated with testicular atrophy. Furthermore, there are no data available on the effects of lindane on testosterone production in Leydig cells or on dose-response relationships between testicular atrophy in rats treated with lindane, serum testosterone levels, and effects on hepatic 2u-globulin synthesis. In spite of this limitation, Dietrich did not hesitate to conclude why this 2u-binding compound did not induce kidney tumors: "Based on the assumption that high levels of 2u-globulin could not have been maintained in male rats treated with high doses of lindane in the 2-year bioassay, due to testicular atrophy and thus a lack of testosterone, it becomes obvious why renal tumors did not develop in lindane-treated male rats." We believe that "obvious conclusions" should be based on data rather than assumptions. Indeed, alternative explanations (e.g., 2u-globulin accumulation is not predictive of kidney tumor response) are compatible with available data and should be tested experimentally.

Third, Dietrich has misrepresented the findings of Green et al. (11) on the influence of 2u-globulin accumulation in the renal carcinogenicity of perchloroethylene in male rats. In an inhalation study of perchloroethylene by the National Toxicology Program, renal tubular cell neoplasms were induced in male rats exposed to 200 or 400 ppm (12). In their studies on the mechanisms of tumor induction by perchloroethylene, Green et al. (11) reported that "this response [accumulation of protein droplets ( 2u-globulin) in the P2 segment of the kidney proximal tubules] was not seen after inhalation exposure to 400 ppm perchloroethylene for 28 days and hence may not be associated with the tumors seen at this dose level." Rather than noting the actual findings of this study, Dietrich chose to exaggerate Green's inferences by claiming that "the tumors observed in the male rat kidney appear to be related to a combination of at least two mechanisms: genotoxicity from the �-lyase pathway and the 2u-globulin mechanism." This is a scientifically unjustified conclusion.

Has the 2u-globulin hypothesis been "meticulously proven" as claimed by Dietrich? One attempt to develop a dosimetry model based on the 2u-globulin hypothesis and which includes synthesis, excretion, degradation, and renal accumulation of 2u-globulin has been reported (13). However, this model was not able to simulate the renal accumulation of 2u-globulin in male rats dosed with 2,4,4-trimethyl-2-pentanol, even when the renal degradation rate of the 2u-ligand complex was assumed to be inhibited beyond what has been reported in the literature [100% inhibition rather than the 30% inhibition reported by Lehman-McKeeman et al. (14)]. Because this test of the 2u-globulin hypothesis (the only one performed to date) could not reproduce the critical quantitative relationships between exposure to an 2u-binding ligand and 2u-globulin accumulation observed in the male rat kidney, development of alternative hypotheses based on the same available information is necessary. Interestingly, Dietrich overlooked this important study by Borghoff et al. (13) in citing data to support the 2u-globulin hypothesis. We agree with the views of de la Iglesia et al. (15) that "clearly new data, rather than new assumptions, will give fresh insight to our understanding of the 2u nephropathy puzzle."

Dietrich and others (16) take the position of declaring that conclusions by original investigators should be accepted as valid, whereas alternative interpretations of the same data by other scientists (17) are distortions of fact. Unfortunately, incorrect speculations by original investigators are not rare. When this possibility exists and the consequences of misinterpretation can adversely affect public health, then it is incumbent on responsible scientists to present their perspectives. For those who care about public health, this is good enough reason to question the validity of "accepted" mechanisms. Furthermore, scientific advancement is based on the principle of rigorous hypothesis testing. To accept and promote unproven hypotheses as fact is equivalent to elevating hypotheses to the level of actual data. This is harmful for the advancement of science. Contrary to the view of Ashby (18), we hold firm that there can be no compromise on this issue. One serious result of uncritical acceptance of hypotheses is that experiments aimed at identifying mechanistic details may not be performed. A consequence of this practice is to denigrate toxicology as a scientific discipline.

Ronald L. Melnick
Michael C. Kohn
James Huff
National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina

References

1. Ashby J. 2�-Globulin nephropathy in white ravens [letter]. Environ Health Perspect 104:1264 (1996).

2. Huff J. Response: 2�-globulin nephropathy, posed mechanisms, and white ravens [letter]. Environ Health Perspect 104:1264-1267 (1996).

3. Dietrich DR. Doubting nongenotoxic mechanisms of renal cancer: comparing apples and oranges in the 2u-globulin hypothesis [letter]. Environ Health Perspect 105:898-902 (1997).

4. Short BG, Bumett VL, Swenberg JA. Elevated proliferation of proximal tubule cells and localization of accumulated 2u-globulin in F344 rats during chronic exposure to unleaded gasoline or 2,2,4-trimethylpentane. Toxicol Appl Pharmacol 101:414-431 (1989).

5. U.S. EPA. Alpha-2U-Globulin: Association with Chemically-Induced Renal Toxicity and Neoplasia in the Male Rat. EPA/625/3-91/019F. Washington, DC:U.S. Environmental Protection Agency, 1991.

6. Partanen T, Heikkila P, Hemberg S, Kauppinen T, Moneta G, Ojajarvi A. Renal cancer and occupational exposure to chemical agents. Scand J Work Environ Health 17:231-239 (1991).

7. Lynge E, Andersen A, Nilsson R, Barlow L, Pukkala E, Nordlinder R, Boffetta P, Grandjean P, Heikkil� P, H�rte LG. Risk of cancer and exposure to gasoline vapors. Am J Epidemiol 145:449-458 (1997).

8. Dietrich DR, Swenberg JA. Lindane induces nephropathy and renal accumulation of 2u-globulin in male but not in female Fischer 344 rats or male NBR rats. Toxicol Lett 53:179-181 (1990).

9. National Cancer Institute. Bioassay of Lindane for Possible Carcinogenicity (CAS No. 58-89-9). NCI Technical Report No 14. Bethesda, MD:National Institutes of Health, 1977.

10. Reuber MD. Carcinogenicity of lindane. Environ Res 19:460-481 (1979).

11. Green R, Odum J, Nash JA, Foster JR. Perchloroethylene-induced rat kidney tumors: an investigation of the mechanisms involved and their relevance to humans. Toxicol Appl Pharmacol 103:77-89 (1990).

12. NTP. Toxicology and Carcinogenesis Studies of Tetrachloroethylene (Perchloroethylene) (CAS No. 127-18-4) in F344/N Rats and B6C3F1 mice (Inhalation Studies). TR 311. Research Triangle Park, NC:National Toxicology Program, 1986.

13. Borghoff SJ, Gargas ML, Andersen ME, Conolly RB. Development of a mechanism-based dosimetry model for 2,4,4-trimethyl-2-pentanol-induced 2u-globulin nephropathy in male Fischer 344 rats. Fundam Appl Toxicol 25:124-137 (1995).

14. Lehman-McKeeman LD, Rivera-Torres MI, Caudill D. Lysosomal degradation of 2uglobulin and 2u-globulin-xenobiotic conjugates. Toxicol Appl Pharmacol 103:539-548 (1990).

15. de la Iglesia FA, Gough AW, Sigler RE. 2u-Globulin nephropathy and ravens: do ravens of a different feather flock together? [letter]. Environ Health Perspect 105:903-904 (1997).

16. Borghoff SJ, Lehman-McKeeman LD, Short BG, Hard GC, Swenberg JA. Critique of R. Melnick's "An alternative hypothesis on the role of chemically induced protein droplet ( 2uglobulin) nephropathy in renal carcinogenesis" [comment]. Regul Toxicol Pharmacol 18:357-364 (1993).

17. Melnick RL. An alternative hypothesis on the role of chemically induced protein droplet ( 2u-globulin) nephropathy in renal carcinogenesis. Regul Toxicol Pharmacol 16:111-125 (1992).

18. Ashby J. The relevance of mechanistic data to the interpretation and extrapolation to humans of rodent carcinogenicity data [letter]. Environ Health Perspect 105:902-903 (1997).

Last Update: October 6, 1997