Pennyroyal Toxicity: Measurement of Toxic Metabolite Levels in Two Cases and Review of the Literature

Methods

Quantification of Pulegone and Menthofuran

Plasma samples were acidified and extracted with diethylether after internal standards were added. To identify and quantify pulegone and menthofuran [14, 15], we compared their gas chromatographic retention times and mass spectra with those of known standards.

Gas chromatographic analysis was done on a Hewlett-Packard (Palo Alto, California) Model 5980. Chromatography was done on a 30 m × 0.320 mm Wall Coated Open Tubular DB-5 fused silica capillary column (J & W Scientific, Folsom, California). Electron-impact mass spectrometry was done using a VG-7070H double-focusing instrument (Manchester, United Kingdom) that was equipped with a Hewlett-Packard Model 5980 Series II gas chromatograph and was electronically linked to a Mass Spectrometry Service data system (Manchester, United Kingdom).

Identification of Protein-Bound Pennyroyal Metabolites by Western Blot Analysis

Liver microsomes were prepared from a liver sample obtained from patient 1 and from a human liver sample obtained from the University of Washington School of Pharmacy Liver Bank. To provide a positive control, a portion of the latter sample was incubated with menthofuran and an NADPH (reduced nicotinamide adenine dinucleotide phosphate)-regenerating system.

Microsomal proteins were subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, and the protein bands were transferred to nitrocellulose solid support. Protein adducts were detected using a primary antibody obtained from rabbits immunized with chemically synthesized oxidative metabolites of menthofuran coupled to metallothionein and with a secondary antibody of horseradish peroxidase-conjugated goat antirabbit IgG (Pierce Chemical Co., Rockford, Illinois). The membranes were developed using 0.004% nickel chloride, 0.0075% hydrogen peroxide, and 0.05% 3,3-diaminobenzidine tetrahydrochloride.

Poison Center Reporting

We selected patients by reviewing all cases involving pennyroyal ingestion for which the San Francisco Bay Area Regional Poison Control Center was primarily consulted during a 2-year period. We included all medically treated or symptomatic cases and excluded two other cases initially reported to a collaborating regional poison control center for which our service provided a secondary consultation. All data were collected by telephone and were recorded on a standard American Association Poison Control Center Cooperative Poison Center report form at the time of the initial consultation. The poison information specialist obtained all pertinent information available. Follow-up contact by telephone was continued until the clinical outcome was determined. The San Francisco Poison Control Center receives an average of 60 000 consultations each year [16].

Literature Review

We identified case reports by searching MEDLINE and Index Medicus and by reviewing the reference citations of all available publications. One reference citation was supposedly a case report of pennyroyal poisoning; however, we later reviewed the original report and discovered that the case did not involve poisoning and appears to have been cited in error [17]. Only 7 of the 18 cases are from the modern medical literature; the others were reported before 1905. The cases reported before 1905 are less well documented, but we did not exclude cases for this reason.

Case Reports

Case 1

A 24-year-old woman repeatedly ingested pennyroyal herbal extract (pennyroyal herb, 48% to 56% in an alcohol base) and black cohosh root (Cimicifuga racemosa) extract for 2 weeks in an attempt to induce an abortion. When this was unsuccessful, she ingested additional unknown amounts of pennyroyal herbal extract and black cohosh root extract over a short period. Soon after, abdominal cramps, chills, vomiting, and syncope developed, and the patient had difficulty walking. She was placed in a cold bath 7 hours after the acute ingestion and began to manifest rigors that her roommate interpreted as a seizure. Paramedics found the patient in cardiopulmonary arrest at a time estimated to be 7.5 hours after the acute ingestion. The patient was intubated, and then cardiopulmonary resuscitation was initiated and continued for 22 minutes until the patient arrived at the hospital. On arrival at the emergency department, the patient's heart rate was 120 beats/min and her blood pressure was 70/40 mm Hg while she received maximal dopamine. Her pupils were fixed and dilated. The physical examination showed coma and a rigid abdomen. A computed tomographic scan of the abdomen suggested a possible ruptured ectopic pregnancy.

Initial laboratory values were the following: sodium level, 157 mmol/L; potassium level, 5.7 mmol/L; chlorine level, 107 mmol/L; bicarbonate level, 8 mmol/L; blood urea nitrogen level, 5.0 mmol/L; creatinine level, 221 µmol/L; glucose level, 6.8 mmol/L; lactic acid level, 21.0 mmol/L; leukocyte count, 37.3 × 10⁹/L; hemoglobin level, 89 g/L; hematocrit, 0.28; platelet count, 256 × 10⁹/L; albumin level, 27 g/L; total bilirubin level, 5.1 µmol/L; aspartate aminotransferase level, 0.82 µkat/L; lactate dehydrogenase level, 3.54 µkat/L; amylase level, 2.35 µkat/L; prothrombin time, 23 seconds; partial prothrombin time, 68 seconds; and international normalized ratio, 4.1. A quantitative plasma human chorionic gonadotropin level indicated that the patient was 1 to 3 months pregnant. Arterial blood gas values (measured while the patient received 100% O₂ by endotracheal tube) were the following: pH, 6.61; PCO ₂, 23 mm Hg; and PO ₂, 503 mm Hg. A toxicology screen was negative for alcohol, acetaminophen, and salicylates.

Laboratory values 36 hours after the acute ingestion were notable for the aspartate aminotransferase level (44.53 µkat/L), the alanine aminotransferase level (29.12 µkat/L), and the lactate dehydrogenase level (68.18 µkat/L). Throughout the initial 12 hours of hospitalization, the patient's course was marked by hemodynamic shock, decreasing hematocrit, and a clinical picture consistent with disseminated intravascular coagulation.

During hospitalization, the patient received 10 units of packed red blood cells and multiple units of fresh frozen plasma. Exploratory laparotomy showed a hemorrhagic, right-sided ectopic pregnancy with indications of superinfection. A substantial amount of old blood, not otherwise quantified, was found. The pregnancy was not ruptured, but there was evidence of bleeding from the end of the tube. No active bleeding was seen during surgery.

A computed tomographic scan of the head was consistent with anoxic encephalopathy. The patient remained unresponsive to all stimuli. Life support was withdrawn, and the patient died 46 hours after the acute pennyroyal ingestion.

Other than the anticipated brain and uterine findings, the most notable abnormalities at autopsy were found in the liver. The substantial centrilobular degeneration and necrosis of the hepatic cells were consistent with a specific toxic insult. Diffuse degenerative changes involving the proximal tubules of the kidney were also noted. The pathologist concluded that the cause of death was multiorgan failure and anoxic encephalopathy secondary to ingestion of pennyroyal and black cohosh. Blood from the heart, collected at autopsy 26 hours after death, was tested for the presence of pulegone and menthofuran. The pennyroyal and black cohosh herbal extracts that the patient had ingested were also tested for the presence of pulegone and menthofuran (Table 1).

Postmortem liver samples were tested for the presence of menthofuran and its metabolites; Western blotting was used as a qualitative test to show adduction of reactive menthofuran metabolites to microsomal proteins. Western blot results were positive for the presence of protein-bound menthofuran metabolites (Figure 2).

Case 2

A 22-month-old, 10-kg girl ingested an unknown amount of pennyroyal oil from a 30-mL bottle. Gastrointestinal decontamination was not done at home. The child arrived at the emergency department 15 minutes after ingestion, and her vital signs were the following: heart rate, 150 beats/min; blood pressure, 104/55 mm Hg; respiratory rate, 30 breaths/min; and temperature, 36.3 °C. The rest of the physical examination was unremarkable, and no gastrointestinal upset was noted. Gastric lavage was done within 30 minutes of ingestion and was followed by administration of activated charcoal (1 g/kg of body weight) and sorbitol (2 mL/kg). The odor of the gastric aspirate was consistent with that of pennyroyal oil. Immediately after administration of gastrointestinal lavage and charcoal and sorbitol, the child received oral N-acetylcysteine, 190 mg/kg, followed by 70 mg/kg every 4 hours (total, 17 doses). This is the standard protocol for acetaminophen overdose [18], adjusted for the coadministration of charcoal.

Three serum samples were collected and frozen at − 20 °C for later menthofuran analysis; the earliest sample was obtained 10 hours after ingestion (see the Methods section). All three samples tested positive for menthofuran, a toxic metabolite of pulegone. The quantitative menthofuran serum level 10 hours after ingestion was 40 ng/mL. Laboratory findings 40 hours after ingestion were the following: alkaline phosphatase level, 2.1 µkat/L; aspartate aminotransferase level, 0.37 µkat/L; prothrombin time, 13 seconds; and partial prothrombin time, 29 seconds (all variables were within normal limits). The patient's course in the hospital was unremarkable, and the patient was discharged to her home in good health.

Case 3

An adult woman of unknown age drank a cup of pennyroyal tea to induce menses. She brewed 1 teaspoon of pennyroyal leaves in a cup of boiling water. Fifteen minutes after ingestion, the patient contacted the San Francisco Poison Control Center reporting dizziness, weakness, a feeling of impending syncope, and no gastrointestinal symptoms. She was seen at a hospital emergency department within 30 minutes of this telephone call. Within 1 hour, her symptoms improved. No laboratory data were obtained. At 24 hours, the patient reported no persistent symptoms.

Case 4

A 24-year-old woman drank two glasses of pennyroyal tea to induce menses. She had learned in an “herbal book” that pennyroyal has been used both as a nonrecommended emmenagogue (agent to induce menses) and as an abortifacient.

The tea was prepared by steeping 2 teaspoons of pennyroyal leaves in 1 pint of hot water for 5 to 10 minutes. Within 15 minutes of drinking the entire amount, the patient developed mild abdominal cramping, but menses did not start. Thirteen hours later, the patient again prepared a cup of pennyroyal tea, this time allowing it to steep for 20 minutes before drinking it. Within 30 minutes, nausea and severe abdominal cramping developed. The abdominal cramping persisted for 4 days, at which time menses began. Five days after the initial ingestion, the patient had no residual symptoms.

Previous Case Reports in the Medical Literature

Through our literature review, we identified 18 reported cases of pennyroyal ingestion (Table 2 and Table 3). All cases occurred in adult women, most of whom had used pennyroyal as an abortifacient. Most of the women ingested pennyroyal oil. Other preparations included a tablet, a tea, essence of pennyroyal (an alcohol-based preparation of the herb), and, in one case, pennyroyal leaves. Patients typically developed gastrointestinal upset and central nervous system effects within 1 to 2 hours after ingestion [9-11, 19-27]. In four cases, central nervous system toxicity included seizures [25, 27-29]. The reported seizures had quick onset and usually manifested within the first 3 hours after ingestion. Pupilary changes have varied. Miotic [21, 29], mydriatic [20, 22, 24-26, 30], and normal [19, 23, 27] pupils have been reported.

Five mL of pennyroyal oil has been associated with coma and seizures (in cases reported before 1905 [25, 27]). However, in all but these two cases, 10 mL or less of pennyroyal oil in adults was generally associated primarily with gastritis and mild central nervous system toxicity but not with hepatic or renal toxicity [9, 10, 22, 24]. In contrast, 15 mL of pennyroyal oil or more ingested by an adult has been fatal [9-11]. In this context, it is important to note that Buechel and colleagues' patient [19], who received prompt N-acetylcysteine treatment after ingesting pennyroyal tea repeatedly over 3 weeks and then drinking 15 mL of pennyroyal oil in a short period, developed no clinical or laboratory evidence of hepatic necrosis. However, other researchers have described patients who allegedly ingested 30 mL of pennyroyal oil and survived [29, 30]. Both of these patients received an emetic agent after ingesting pennyroyal.

None of the 15 patients who survived had documented signs of liver injury, but few of these patients were evaluated for subclinical impairment, such as elevated liver enzyme levels. Of the three patients who died, one had extensive centrilobular hepatic necrosis [9, 10], another had slight centrilobular hepatic cloudy swelling [8], and the third (described in 1897) had poorly documented pathologic findings [11]. The two patients described in the modern medical literature who died with evidence of hepatic injury also had evidence of renal injury [8-10]. In these cases, clinical and laboratory evidence of the hepatic and renal injury was noted within 24 hours after acute ingestion. Disseminated intravascular coagulation was reported in one case approximately 40 hours after acute ingestion [9, 10].

Discussion

Case Findings

The patient in case 1 developed multiorgan failure and died after ingesting two herbs: pennyroyal and black cohosh. The primary effects associated with black cohosh ingestion are gastrointestinal upset, dizziness, and headache [31-33]. Neither hepatotoxicity nor multiorgan failure has been reported with black cohosh ingestion alone. The centrilobular hepatic necrosis observed in this case may simply have been caused by prolonged shock, but it is also consistent with pennyroyal toxicity. The Western blot results, which indicated the presence of menthofuran metabolite protein adducts in the liver, are consistent with pennyroyal ingestion as a cause of the hepatic necrosis. As shown in Table 2, other reported cases of pennyroyal ingestion were associated with depressed mental status and multiorgan effects not directly attributed to hepatic toxicity. In fatal cases of pennyroyal toxicity, the liver is probably not the only target organ for toxicity.

Case 2 highlights the clinical management questions raised by our current understanding of pennyroyal metabolism. The decision to treat the patient with N-acetylcysteine was made on the basis of a potentially fatal ingestion of a toxin that may form a reactive electrophilic metabolite, the fact that serum levels are not readily available or interpretable, and the proposed antidotal efficacy of N-acetylcysteine without major toxicity. Fortunately, the patient had no ill effects, possibly because of prompt gastrointestinal cleansing, the altered metabolism of a child, treatment with N-acetylcysteine, or a nontoxic ingested amount of pennyroyal. Young children have greater tolerance of acetaminophen toxicity without glutathione replenishment, possibly because of increased detoxication by sulfation [34, 35]. It is unknown whether this or other protective mechanisms exist in children for pennyroyal ingestion.

We have included cases 3 and 4 not because they involved any serious adverse effects but because they show a common scenario that raises substantial therapeutic management issues. As ascertained by history, the young women who had these cases had ingested amounts of pennyroyal thought to be minimally toxic, but they had symptoms that were of concern. These cases raise the question of the indication for treatment with N-acetylcysteine, even when the history suggests minor ingestion. The decision not to treat was made on the basis of the knowledge that previous patients had ingested similar amounts of pennyroyal and survived without any serious sequelae. The lack of gastrointestinal symptoms in case 3 and the lack of central nervous system effects in case 4 also suggest minor ingestion.

Pennyroyal oil ingestion is treated by prompt, aggressive cleansing of the stomach with gastric lavage and activated charcoal for patients evaluated soon after ingestion. Emesis with ipecac is less desirable because of the rapid absorption of pennyroyal oil, the risk for aspiration pneumonitis, and the potential for rapid onset of central nervous system effects. On the basis of research by Thomassen and colleagues [36] and Gordon and colleagues [37], Buechel and colleagues' case report [19], and the relatively low toxicity of N-acetylcysteine [38], we recommend that N-acetylcysteine be administered as soon as possible after all pennyroyal overdoses that are in the toxic range (> 10 mL of pennyroyal oil). The dosing regimen of N-acetylcysteine for pennyroyal overdose is still empiric. It is reasonable to administer a loading dose of N-acetylcysteine, 140 mg/kg, and continue therapy with 70 mg/kg every 4 hours. The necessary duration of N-acetylcysteine therapy is unknown. Because the half-life of pennyroyal metabolites in animals is only a few hours, maintenance doses for 24 hours should be adequate. Care is supportive, with close attention to mental status, respiratory status, and hepatic and renal function. Laboratory studies should include monitoring of coagulation and hepatic and renal function.

Metabolism and Toxicity of Pennyroyal Oil

Pennyroyal oil is a typical mint oil that contains several monoterpenes. One of these monoterpenes, (R)-(+)-pulegone, is found in greater amounts in pennyroyal than in other mint oils. On the basis of its known toxic effects on the liver [37, 39] and lung [37] and its ability to cause convulsions at higher doses [37], this monoterpene is thought to be largely responsible for the organ toxic effects of pennyroyal oil in small rodents. Studies have shown that oxidative metabolites of pulegone cause the organ toxic effects of this monoterpene because inhibitors of cytochromes P450 mitigate the organ damage [14, 36, 37, 40]. Moreover, the major proximate hepatotoxic metabolite of pulegone has been identified as menthofuran, which is then further oxidized by cytochromes P450 until it ultimately becomes a hepatotoxin [3, 14, 40-43]. However, pulegone is oxidized to several other metabolites that may also be involved in causing organ toxic effects [3, 36, 42, 44, 45]. Reactive oxidative metabolites of pulegone and menthofuran bind to target cell proteins, and the extent of this binding parallels the extent of cellular damage [14, 40, 46].

Role of Glutathione

Although depletion of the sulfhydryl-containing tripeptide, glutathione, markedly increases the extent of hepatotoxicity caused by pulegone, it only marginally affects the toxicity caused by menthofuran [36]. Similarly, pulegone forms reactive metabolites that deplete hepatic glutathione levels, whereas menthofuran only modestly depletes hepatocellular glutathione levels [36, 37]. Depletion of glutathione levels by pulegone appears to be related to the formation of electrophilic reactive metabolites of pulegone that form covalent adducts with the nucleophilic cysteinyl sulfhydryl group of glutathione [45]. Thus, the use of N-acetylcysteine to protect against pennyroyal oil toxicity is empiric and, on the basis of the results of the animal studies described above, would only be beneficial within the first few hours of pennyroyal poisoning. However, N-acetylcysteine may protect cells from damage by more than one mechanism that may work in later stages of cell injury.

Kinetics of Toxic Doses of Pulegone

Kinetic studies on pulegone have only been done in rats [42]. A single 150 mg/kg intraperitoneal dose of pulegone yielded peak pulegone plasma levels of 13.5 ± 3.0 µg/mL at approximately 15 minutes and had a terminal half-life of about 1 hour. This pulegone dose resulted in peak menthofuran plasma levels of 7.0 ± 1.2 µg/mL at approximately 1 hour and had a terminal half-life of about 2 hours.

The serum levels detected in our patients are the first to be reported for humans. They raise several interesting points but should be interpreted with caution. In case 1, serum samples collected at autopsy 26 hours after death (72 hours after the acute ingestion) yielded 18 ng of pulegone per mL and 1 ng of menthofuran per mL. The 10-hour menthofuran serum level in case 2 was 40 ng/mL, but no pulegone was detected in this sample. No other human serum levels were available for comparison.

In animals, the half-life of menthofuran is 2.2 hours [36]. If we assume first-order kinetics, the peak menthofuran serum level 1 hour after ingestion yields an estimated range of 300 to 400 ng/mL in case 2. In animals, peak serum levels of menthofuran of 2250 ng/mL at 1 hour resulted in only moderate hepatotoxicity. If we assume that the human dose-response curve for toxicity is similar to that of small rodents, then case 2 would have had a good outcome regardless of N-acetylcysteine administration (that is, the exposure was not in the toxic range).

Compared with animal data, the pulegone level in case 1 is extremely high, but this level may be falsely elevated because of postmortem cellular shifts. The relative pulegone and menthofuran levels in this case are interesting. In rats and mice, pulegone is metabolized to menthofuran. This patient had an elevated pulegone level and a low menthofuran level despite the time elapsed for metabolism. On the basis of experimental animal models, we would have expected a higher menthofuran level relative to the pulegone level so long after ingestion. Metabolism in humans may differ in that menthofuran is not a major metabolite, or it may have greatly altered kinetics in the toxic dosing range. If this is true, N-acetylcysteine would play an even greater role in treating the overdose, because menthofuran is probably not detoxified by N-acetylcysteine, but pulegone is. Nonetheless, we cannot exclude other explanations for the observed low menthofuran level relative to the pulegone level, including sampling effects or a postmortem distribution artifact.

In conclusion, pennyroyal is a widely available herbal product that has proved fatal after relatively small ingestions. We have reported four cases that occurred at one poison control center during a 2-year period. It is important to note that the national incidence of pennyroyal use is unknown. These cases were collected from the San Francisco Bay area, which may not be representative of the rest of the United States. Two of the cases include the first reported human serum levels of pulegone and menthofuran. One patient died, one received N-acetylcysteine, and two had mild exposures to pennyroyal. These cases represent the spectrum of damage caused by this toxin. Evidence from animal experiments suggests that N-acetylcysteine provides at least partial protection from the hepatotoxicity caused by pennyroyal, emphasizing the role of early intervention in severe cases. Its wide commercial availability and reputation as an abortifacient continue to make pennyroyal a serious public health concern.

Dr. Meeker: Institute of Forensic Sciences, Toxicology Laboratory, 2945 Webster Street, Oakland, CA 94609.

Mr. Khojateh-Bakht and Drs. Oishi and Nelson: University of Washington, Department of Medicinal Chemistry, Box 357610, Seattle, WA 98195-7610.

Dr. Blanc: Division of Occupational and Environmental Medicine, University of California, Box 0924, San Francisco, CA 94143.