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PubMed1999

Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (-)-4S-limonene-6-hydroxylase.

The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In peppermint (Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol. The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the C6-hydroxylase by functional expression using the baculovirus-Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene-3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants.

Amino Acid Sequence↗
PubMed1989

d-Limonene-induced male rat-specific nephrotoxicity: evaluation of the association between d-limonene and alpha 2u-globulin.

d-Limonene is a naturally occurring monoterpene, which when dosed orally, causes a male rat-specific nephrotoxicity manifested acutely as the exacerbation of protein droplets in proximal tubule cells. Experiments were conducted to examine the retention of [14C]d-limonene in male and female rat kidney, to determine whether d-limonene or one or more of its metabolites associates with the male rat-specific protein, alpha 2u-globulin, and if so, to identify the bound material. The results indicated that, 24 hr after oral administration of 3 mmol d-limonene/kg, the renal concentration of d-limonene equivalents was approximately 2.5 times higher in male rats than in female rats. Equilibrium dialysis in the presence or absence of sodium dodecyl sulfate indicated that approximately 40% of the d-limonene equivalents in male rat kidney associated with proteins in a reversible manner, whereas no significant association was observed between d-limonene equivalents and female rat kidney proteins. Association between d-limonene and male rat kidney proteins was characterized by high-performance gel filtration and reverse-phase chromatography. Gel filtration HPLC indicated that d-limonene in male rat kidney is associated with a protein fraction having a molecular weight of approximately 20,000. Separation of alpha 2u-globulin from other kidney proteins by reverse-phase HPLC indicated that d-limonene associated with a protein present only in male rat kidney which was definitively identified as alpha 2u-globulin by amino acid sequencing. The major metabolite associated with alpha 2u-globulin was d-limonene-1,2-oxide. Parent d-limonene was also identified as a minor component in the alpha 2u-globulin fraction. Thus, d-limonene, and more specifically d-limonene-1,2-oxide, associates with alpha 2u-globulin in a reversible manner in male rat kidney. This interaction may be responsible for excessive accumulation of alpha 2u-globulin in kidneys of male rats exposed to d-limonene.

Administration, Oral↗
PubMed2000

Cloning and expression of the limonene hydroxylase of Bacillus stearothermophilus BR388 and utilization in two-phase limonene conversions.

A 3.6-kb fragment of Bacillus stearothermophilus BR388 chromosomal DNA that confers growth on limonene to Escherichia coli has been sequenced, revealing a single open reading frame encoding a single subunit limonene hydroxylase containing 444 amino acid residues. This enzyme proved capable of limonene hydroxylation to a mixture of carveol and perillyl alcohol as well as dehydrogenation of these products to carvone and perillyl aldehyde. Oxygen, FAD, and NADH were found to stimulate the hydroxylation reaction in cell extracts, and NAD+ stimulated the dehydrogenase reaction. In two-phase bioconversions using viable E. coli cells over-expressing the limonene hydroxylase, perillyl alcohol and carvone were the principal products observed.

Alcohol Dehydrogenase↗
PubMed1991

Behavior of alpha 2u-globulin accumulating in kidneys of male rats treated with d-limonene: kidney-type alpha 2u-globulin in the urine as a marker of d-limonene nephropathy.

Effects of d-limonene on alpha 2u-globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d-limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type alpha 2u-globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum alpha 2u-globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating alpha 2u-globulin levels were not affected by the d-limonene administration. While the molecular weight of the major alpha 2u-globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type-alpha 2u-globulin was observed in the urine from treated rats. The results were indicative of elimination of alpha 2u-globulin from the kidney to urine, the appearance of kidney-type-alpha 2u-globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the alpha 2u-globulin (kidney-type) in the urine might be used as an indicator of chemically induced alpha 2u-globulin nephropathy.

Alpha-Globulins↗
PubMed1975

Studies on the metabolism of d-Limonene (p-Mentha-1,8-diene). III. Effects of d-Limonene on the lipids and drug-metabolizing enzymes in rat livers.

1. After a single oral dose of d-limonene (200-1200 mg/kg) no effects were observed on liver triglyceride, microsomal protein, cytochrome b5, and the drug-metabolizing enzymes. Glycogen content was slightly decreased at doses higher than 800 mg/kg, and cytochrome P-450 and delta-aminolaevulinic acid synthetase activity was slightly increased at 1200 mg/kg. 2. After repeated treatment (400 mg/kg/day) for 30 days, the relative liver weight and hepatic phospholipid content were only slightly increased, and liver and serum cholesterol were decreased 49 and 8%, respectively. Of the phospholipid fatty acids, palmitic, linoleic and arachidonic acids were increased, and stearic acid was decreased. Aminopyrine demethylase and aniline hydroxylase were increased 26 and 22%, respectively, and cytochrome P-450 and b5 were likewise increased 31 and 30%.

5-Aminolevulinate Synthetase↗
PubMed1999

Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene.

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.

Alcohol Oxidoreductases↗
PubMed1991

The human relevance of the renal tumor-inducing potential of d-limonene in male rats: implications for risk assessment.

The monoterpene d-limonene is a naturally occurring chemical which is the major component in oil of orange. Currently, d-limonene is widely used as a flavor and fragrance and is listed to be generally recognized as safe (GRAS) in food by the Food and Drug Administration (21 CFR 182.60 in the Code of Federal Regulations). Recently, however, d-limonene has been shown to cause a male rat-specific kidney toxicity referred to as hyaline droplet nephropathy. Furthermore, chronic exposure to d-limonene causes a significant incidence of renal tubular tumors exclusively in male rats. Although d-limonene is not carcinogenic in female rats or male and female mice given much higher dosages, the male rat-specific nephrocarcinogenicity of d-limonene may raise some concern regarding the safety of d-limonene for human consumption. A considerable body of scientific data has indicated that the renal toxicity of d-limonene results from the accumulation of a protein, alpha 2u-globulin, in male rat kidney proximal tuble lysosomes. This protein is synthesized exclusively by adult male rats. Other species, including humans, synthesize proteins that share significant homology with alpha 2u-globulin. However, none of these proteins, including the mouse equivalent of alpha 2u-globulin, can produce this toxicity, indicating a unique specificity for alpha 2u-globulin. With chronic exposure to d-limonene, the hyaline droplet nephropathy progresses and the kidney shows tubular cell necrosis, granular cast formation at the corticomedullary junction, and compensatory cell proliferation. Both d-limonene and cis-d-limonene-1,2-oxide (the major metabolite involved in this toxicity) are negative in in vitro mutagenicity screens. Therefore, the toxicity-related renal cell proliferation is believed to be integrally involved in the carcinogenicity of d-limonene as persistent elevations in renal cell proliferation may increase fixation of spontaneously altered DNA or serve to promote spontaneously initiated cells. The scientific data base demonstrates that the tumorigenic activity of d-limonene in male rats is not relevant to humans. The three major lines of evidence supporting the human safety of d-limonene are (1) the male rat specificity of the nephrotoxicity and carcinogenicity; (2) the pivotal role that alpha 2u-globulin plays in the toxicity, as evidenced by the complete lack of toxicity in other species despite the presence of structurally similar proteins; and (3) the lack of genotoxicity of both d-limonene and d-limonene-1,2-oxide, supporting the concept of a nongenotoxic mechanism, namely, sustained renal cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Human metabolism of the experimental cancer therapeutic agent d-limonene.

d-Limonene has efficacy in preclinical models of breast cancer, causing > 80% of carcinomas to regress with little host toxicity. We performed a pilot study on healthy human volunteers to identify plasma metabolites of limonene and to assess the toxicity of supradietary quantities of d-limonene. Seven subjects ingested 100 mg/kg limonene in a custard. Blood was drawn at 0 and 24 h for chemistry-panel analysis and at 0, 4, and 24 h for limonene-metabolite analysis. On-line capillary gas chromatography/mass spectrometry (GC/MS) analysis indicated that at least five compounds were present at 4 h that were not present at time zero. Two major peaks were identified as the rat limonene metabolites dihydroperillic acid and perillic acid, and two minor peaks were found to be the respective methyl esters of these acids. A third major peak was identified as limonene-1,2-diol. Limonene was a minor component. At a dose of 100 mg/kg, limonene caused no gradable toxicity. Limonene is metabolized by humans and rats in a similar manner. These observations and the high therapeutic ratio of limonene in the chemotherapy of rodent cancers suggest that limonene may be an efficacious chemotherapeutic agent for human malignancies.

Adult↗
PubMed1992

Identification of metabolites of the antitumor agent d-limonene capable of inhibiting protein isoprenylation and cell growth.

Limonene has been shown to be an effective, nontoxic chemopreventive and chemotherapeutic agent in chemically induced rat mammary-cancer models. The present study characterized circulating metabolites of limonene in female rats and determined their effects on cell growth. Metabolism of limonene was analyzed in plasma extracts by gas chromatography. Rapid conversion of limonene to two major metabolites was detected. These metabolites comprised more than 80% of the circulating limonene-derived material at 1 h after administration and thereafter, whereas limonene itself accounted for only 15%. The metabolites were characterized by mass spectroscopy and infrared spectroscopy. The probable structures were synthesized, and identities were confirmed by comparison of retention times and mass spectra. The two major circulating metabolites of limonene were found to be perillic acid and dihydroperillic acid. We have previously reported that limonene, perillic acid, and dihydroperillic acid inhibit the posttranslational isoprenylation of p21ras and other 21- to 26-kDa cell-growth-associated proteins in NIH3T3 cells and in mammary epithelial cells. In the present study, perillic acid was found to inhibit cell growth in a dose-dependent manner. Thus, perillic acid and dihydroperillic acid, the two major circulating metabolites of limonene in the rat, are more potent inhibitors of protein isoprenylation than is limonene, and perillic acid is also a more potent inhibitor of cell growth. These data raise the possibility that the antitumor effects of limonene in vivo may be mediated via perillic acid and, perhaps, other metabolites.

3T3 Cells↗
PubMed1989

Acute and subchronic nephrotoxicity of d-limonene in Fischer 344 rats.

In the studies described here, we have examined the sex-specific sensitivity of rat kidney to d-limonene. At 24 hr after an acute dose of 200 mg d-limonene/kg body weight administered to adult male and female Fischer 344 rats by oral gavage, an increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2u-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene. In a separate subchronic study, groups of 5-wk-old male rats were administered d-limonene in a corn oil vehicle at 0 (control), 2, 5, 10, 30, or 75 mg/kg body weight by single daily gavage (5 days/wk) for 13 wk. Rats from selected dose groups received interim necropsies from days 8-29, while all groups were necropsied at the end of the study. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg d-limonene/kg body weight. Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis. The no-observable-effect level for these effects was 5 mg d-limonene/kg body weight. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose. It is concluded that treatment with d-limonene caused an increase in the formation of hyaline droplets in male rats only, that this increase was associated with an accumulation of alpha 2u-globulin, that d-limonene (or its metabolite) accumulated significantly in male rat kidney compared with that in females and that subchronic dosing produced a triad of morphological changes in the male rat kidney. These observations suggest that d-limonene caused nephrotoxicity specific to the male rat and that this toxicity may not be predictive of a similar response in humans.

Acute Kidney Injury↗
PubMed1994

Limonene chemoprevention of mammary carcinoma induction following direct in situ transfer of v-Ha-ras.

Monoterpenes, including limonene and its in vivo rat plasma metabolites, have been shown to be inhibitors of protein isoprenylation of small G proteins, including p21 ras. In addition, dietary limonene has been shown to be capable of preventing the development and causing the regression of chemically induced mammary carcinomas, many of which contain activated ras oncogenes. On the basis of these observations, it was hypothesized that a possible mechanism by which limonene exerts its effects on the chemoprevention and regression of mammary tumors involves the inhibition of protein isoprenylation of the small G protein p21. In the first study, we asked whether dietary limonene was able to prevent the development of mammary carcinomas which were induced using direct retroviral gene transfer of v-Ha-ras into the mammary parenchyma in situ. Limonene modified neither the rate of gene transfer nor the stability of gene expression. However, limonene did greatly inhibit the formation of mammary carcinomas induced by the insertion of activated ras. In a follow-up study, we asked whether chemoprevention by limonene was preferentially effective against a subset of chemically induced mammary carcinomas with activated ras. Rats were fed limonene to prevent the development of N-nitroso-N-methylurea-induced mammary tumors, a majority of which contain the activated Ha-ras oncogene. As expected, limonene administration increased the latency period and lowered the frequency of mammary carcinoma development as compared to controls. However, tumor characterization revealed that limonene treatment did not alter the percentage of carcinomas with activated ras. These studies are consistent with the above studies in that limonene is effective in preventing mammary carcinomas with activated ras. Interestingly, carcinomas without activated ras were prevented to the same extent as those with the activated oncogene.

Animals↗
PubMed1994

d-Limonene induced hyaline droplet nephropathy in alpha 2u-globulin transgenic mice.

d-Limonene is a hyaline droplet inducing agent and produces nephrotoxicity in male rats when the 1,2-epoxide metabolite binds to alpha 2u-globulin. Mice, which do not synthesize alpha 2u-globulin, are resistant to hyaline droplet nephropathy. In this study, the ability of d-limonene to cause hyaline droplet nephropathy in a transgenic mouse engineered to express alpha 2u-globulin was evaluated. The C57BL/6-derived mice excreted 0.4 +/- 0.1 mg alpha 2u-globulin/day, or approximately 16 mg alpha 2u-globulin/kg body wt. This represents about 30% of the amount excreted by adult male rats (11.9 +/- 1.1 mg/day or approximately 48 mg/kg). Transgenic mice excreted less mouse urinary protein (9.3 +/- 1.2 mg/day) than normal mice (15.1 +/- 1.6 mg/day). Unlike normal male rats, untreated transgenic mice did not show significant spontaneous hyaline droplet formation. Liver microsomes from naive transgenic mice oxidized d-limonene to the cis- and transisomers of the 1,2-epoxide, and following oral treatment with [14C]d-limonene reversible binding of d-limonene equivalents to renal cytosolic proteins was observed. Furthermore, with d-limonene treatment, hyaline droplets were observed in the transgenic mouse kidneys. These droplets, however, were much smaller in size than those seen in d-limonene-treated male rats. The accumulation of alpha 2u-globulin in the kidneys of transgenic mice and normal male rats before and after d-limonene treatment was analyzed by Western blotting. These results indicated that alpha 2u-globulin was present in the kidneys of the control transgenic mice, despite the lack of spontaneous hyaline droplet formation. After d-limonene treatment, approximately a three-fold increase in alpha 2u-globulin in the transgenic mouse kidney was observed, a response similar in magnitude to that seen in d-limonene-treated male rats. These results indicate that expression of alpha 2u-globulin in a species that does not normally develop hyaline droplet nephropathy is necessary and sufficient to render that species sensitive to this renal toxicity.

Alpha-Globulins↗
PubMed1992

Air oxidation of d-limonene (the citrus solvent) creates potent allergens.

Products containing as much as 95% of d-limonene are used for, e.g., degreasing metal before industrial painting and for cleaning assemblies. Experimental studies on the sensitizing potential of limonene show diverging results. In a previous study, we found that the sensitizing potential of d-limonene increased with prolonged air exposure. The aim of this study was to make further chemical analyses, to identify compounds formed by air exposure of d-limonene and to study their allergenic potential. d-limonene was found to be a sensitizer after prolonged exposure to air according to 2 Freund's complete adjuvant test (FCAT) experiments and 1 guinea pig maximization test (GPMT) study. No significant response was obtained to d-limonene not air exposed, even if the animals were sensitized to oxidized d-limonene. 5 main oxidation products of d-limonene were identified. (R)-(-)-carvone and a mixture of cis and trans isomers of (+)-limonene oxide were found to be potent sensitizers, while no significant reactions were obtained in the animals induced with a mixture of cis and trans isomers of (-)-carveol. It can be concluded that air oxidation of d-limonene is essential for its sensitizing potential, and that potent allergens are created.

Air Pollutants↗
PubMed1993

Effects of D-limonene on hepatic microsomal monooxygenase activity and paracetamol-induced glutathione depletion in mouse.

1. D-Limonene, a monoterpenoid constituent of citrus fruit oil, blocks tumour induction by chemical carcinogens in laboratory animals, apparently by preventing bioactivation of procarcinogens and by enhancing conjugation of proximal carcinogenic metabolites. 2. Inhibitory effects of D-limonene were measured in vitro using cytochrome P450 isoform-specific substrates. D-Limonene inhibited p-nitrophenol hydroxylase (pNP) activity in vitro in liver microsomes from acetone-, phenobarbital (PB)- and beta-naphthoflavone (BNF)-treated mouse, and 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes from PB- and BNF-treated mouse. p-Nitrophenol and ethoxyresorufin are substrates for cytochromes P2E1 and P1A1, respectively. No inhibition of benzphetamine (BNZP) or aminopyrine (AP) demethylases by D-limonene was observed. 3. EROD, BNZP and AP activities in liver microsomes were increased 18 h after i.p. administration of D-limonene to acetone-induced mouse, while pNP activity was unchanged. The immunodetectable protein level of cytochrome P2B1 in non-acetone treated mouse was increased 18 h after D-limonene, with no differences in P2E1 or P1A1. 4. Acute D-limonene did not protect against paracetamol (acetaminophen)-induced depletion of liver reduced glutathione (GSH). A prolonged paracetamol challenge (0.6% diet for 10 days) elevated liver cytosolic GSH-S-transferase activity (GST) two-fold and decreased liver GSH to 46% of control values. Dietary D-limonene (1.0% diet for 10 days) maintained liver GSH concentrations at 92% of control values in the paracetamol-challenged mouse without altering GST activity. D-Limonene also increased liver GSH concentration (23%) in mouse fed 1.0% D-limonene alone.

Acetaminophen↗
PubMed1994

Risk assessment of d-limonene: an example of male rat-specific renal tumorigens.

The naturally occurring food constituent d-limonene has been found to cause tumors at high doses only in the kidney of the male rat in association with the development of hyaline droplet nephropathy. In contrast, neither kidney tumors nor the associated nephropathy have been found in female rats or mice at much higher doses. Adult male rats produce large quantities of a specific low-molecular-weight protein in the liver, which is known as alpha 2U-globulin (alpha 2U-g). With administration of sufficient doses of d-limonene to male rats, this protein has been found to accumulate excessively in the P2 segment cells of renal proximal tubules, resulting in hyaline droplet formation as a manifestation of protein overload. Hyaline droplet accumulation is the first stage in a unique sequence of nephropathic lesions (also known as alpha 2U-g nephropathy), including granular casts in the outer medulla and linear mineralization in the papilla. The mechanism underlying protein accumulation appears to be the reversible binding of chemical to alpha 2U-g with subsequent prolongation of its half-life in the tubule cell. In the case of d-limonene, the minor metabolite d-limonene-1,2-oxide has been shown to be the primary chemical species that binds reversibly to alpha 2U-g, impeding the normal process of lysosomal proteinase degradation of alpha 2U-g. The ensuing nephropathy is associated with a sustained increase in compensatory renal tubule cell proliferation, which provides the putative mechanistic link with renal tumor formation possibly through tumor promotion of spontaneously initiated cells or enhanced spontaneous mutagenesis. This proposed mechanism has been supported by additional information, including negative genotoxicity tests for d-limonene and its oxide metabolites, experimentally verified tumor promotion, and enhanced cell proliferation primarily in P2 segment tubule cells in male F344 rats, but no such effects in the alpha 2U-g-deficient NBR rat. The mechanism of d-limonene tumor development does not appear to be possible in humans since neither the quantity nor the type of protein that binds d-limonene or d-limonene-1,2-oxide is present. The deduction that the renal tumors induced in male rats are not relevant to human carcinogenicity in the hazard evaluation step of risk assessment completes the evaluation of human risk for d-limonene. Consequently, it can be concluded that d-limonene does not pose any carcinogenic or nephrotoxic risk to humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Selective inhibition of isoprenylation of 21-26-kDa proteins by the anticarcinogen d-limonene and its metabolites.

Limonene has chemotherapeutic activity against chemically induced rat mammary carcinomas, many of which contain activated ras genes. Given the recent discovery of the post-translational modification of p21ras and other cell growth-associated proteins by intermediates in the mevalonic acid pathway, and the common biochemical origins of limonene and these isoprene products, we investigated the effect of limonene on protein isoprenylation. NIH3T3 and human mammary epithelial cells were incubated with lovastatin and [2-14C]mevalonolactone in the absence and presence of limonene. Labeled proteins were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Limonene inhibited isoprenylation of a class of cellular proteins of 21-26 kDa, including p21ras and possibly other small GTP-binding proteins, in a dose-dependent manner in both cell lines. In contrast, limonene did not affect the isoprenylation of several other proteins, including nuclear lamins. Limonene is metabolized extensively in vivo but not in cultured cells. The two major rat serum metabolites of limonene, perillic acid and dihydroperillic acid, were more potent than limonene in the inhibition of isoprenylation. These results demonstrate that limonene selectively inhibits isoprenylation of 21-26-kDa proteins at a point in the mevalonic acid pathway distal to 3-hydroxy-3-methylglutaryl coenzyme A reductase, and they provide a plausible explanation for its chemotherapeutic activity. Inhibition of isoprenylation of proteins such as p21ras and other small GTP-binding proteins would alter their intracellular localization and, hence, disrupt their biological activity.

Animals↗
PubMed1998

Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases.

An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50 degrees C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4'-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1, 10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.

Amino Acid Sequence↗